Compositions, systems, and methods for cell differentiation using targeted gene activation of DLL4 and / or VCAM1

Abstract

Provided are DNA-targeting systems, such as CRISPR-Cas / guide RNA (gRNA) systems, that bind to or target a target site in a DLL4 gene or a VCAM1 gene. In some embodiments, the provided DNA-targeting systems promote transcriptional activation of such genes to promote differentiation of hematopoietic progenitor cells into a differentiated population of cells, such as lymphoid progenitor or lymphoid cells. Also provided herein are methods and uses related to the provided compositions, for example in facilitating lymphoid progenitor cell differentiation.

Description

COMPOSITIONS, SYSTEMS, AND METHODS FOR CELL DIFFERENTIATION USING TARGETED GENE ACTIVATION OF DLL4 AND / OR VCAM1Cross-Reference to Related Applications

[0001] This application claims priority from U.S. provisional application No. 63 / 669,223 filed July 9, 2024, and U.S. provisional application No. 63 / 823,717 filed June 13, 2025, the contents of each of which are incorporated by reference in their entireties.Incorporation by Reference of Sequence Listing

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 224742003740SeqList. xml, created on July 8, 2025, which is 377,112 bytes in size. The information in the electronic format of the Sequence Listing is herein incorporated by reference in its entirety.Field

[0003] The present disclosure relates in some aspects to DNA-targeting systems, such as CRISPR-Cas / guide RNA (gRNA) systems, that bind to or target a target site in a DLL4 gene or a VCAM1 gene. In some aspects, the provided DNA-targeting systems of the present disclosure promote transcriptional activation of such genes to promote differentiation of hematopoietic progenitor cells into a differentiated population of cells, such as lymphoid progenitor or lymphoid cells. In some aspects, the present disclosure is directed to methods and uses related to the provided compositions, for example in facilitating lymphoid progenitor cell differentiation.Background

[0004] The ability to modulate the Notch signaling pathway is the foundation for producing highly functional cells for immunotherapy. However, current methods for differentiating stem cells into lymphoid progenitor cells (e.g., common lymphoid progenitors; CLPs) or lymphoid cells rely on the provision of external Notch- signaling ligands, which is laborious, costly, and difficult to scale. Therefore, there is a need for new and improved methods to overcome these challenges. The present disclosure addresses these and other needs.Summary

[0005] Provided herein is a DNA-targeting system comprising a DNA-targeting modules, wherein the DNA-targeting module comprises: (a) a DNA-targeting module for increasing transcription of a DLL4 gene, wherein the DNA-targeting module comprises a fusion protein comprising (i) a DNA-binding domain that binds to a target site for DLL4, and (ii) at least one transcriptional activator effector domain; or (b) a DNA-targeting module for increasing transcription of a VCAM1 gene, wherein the DNA-targeting module comprises a fusion protein comprising (i) a DNA-binding domain that binds to a target site for VCAM1, and (ii) at least one transcriptional activator effector domain. In some embodiments, the DNA-targeting system comprises a plurality of DNA-targeting modules that include at least one DNA-targeting module of (a) for increasing transcription of a DLL4 gene and / or of (b) for increasing transcription of a VCAM1 gene. In some embodiments, the plurality of DNA-targeting modules include a DNA- targeting module of (a) for increasing transcription of a DLL4 gene and of (b) for increasing transcription of a VCAM1 gene.

[0006] Also provided herein is a DNA-targeting system comprising a plurality of DNA- targeting modules, wherein the plurality of DNA-targeting modules comprises: (a) a first DNA- targeting module for increasing transcription of a DLL4 gene, wherein the first DNA-targeting module comprises a fusion protein comprising (i) a DNA-binding domain that binds to a target site for DLL4, and (ii) at least one transcriptional activator effector domain; and (b) a second DNA-targeting module for increasing transcription of a VC AMI gene, wherein the second DNA-targeting module comprises a fusion protein comprising (i) a DNA-binding domain that binds to a target site for VCAM1, and (ii) at least one transcriptional activator effector domain.

[0007] In some of any embodiments, the target site for DLL4 is in the gene or a regulatory DNA element thereof. In some embodiments, the regulatory DNA element is an enhancer or a promoter of the gene. In some embodiments, the regulatory DNA element is a promoter of the gene.

[0008] In some of any embodiments, the target site for DLL4 is within the genomic coordinates chrl5:40,905,000 to chrl5:40,943,500. In some of any embodiments, the target site for DLL4 is within the genomic coordinates chrl5: 40,905,000 to chrl 5:40,909,000; chrl5: 40,925,250 to chrl5:40,934,00; chrl5: 40,937,800 to chrl5:40,938,800; or chrl5: 40,940,000 to chr!5:40,943,500. In some of any embodiments, the target site for DLL4 is within the genomiccoordinates chrl5: 40,925,250 to chrl5:40,934,00. In some of any embodiments, the target site for DLL4 is within the genomic coordinates chrl5: 40,928,340 to chrl5:40,930,340.

[0009] In some of any embodiments, the target site for DLL4 is within 1000 base pairs of the transcription start site (TSS) of the gene. In some of any embodiments, the target site for DLL4 is within 20 base pairs, 50 base pairs, 100 base pairs, 200 base pairs, 300 base pairs, 400 base pairs, 500 base pairs, 600 base pairs, or any value between any of the foregoing, of the TSS of the gene.In some of any embodiments, the target site for DLL4 is within 550 base pairs upstream of the TSS of the gene. In some of any embodiments, the target site for DLL4 is within 250 base pairs upstream of the TSS of the gene. In some of any embodiments, the target site for DLL4 is within the genomic coordinates chrl5: 40,929,100 to chrl5:40,929,170.

[0010] In some of any embodiments, the target site for DLL4 is a target site having the sequence set forth in any one of SEQ ID NOs:l, 2 or 147-166, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any embodiments, the target site for DLL4 is within the genomic coordinates chrl5:38,734,103 to chrl5:38,736,163. In some of any embodiments, the target site for DLL4 is a target site having the sequence set forth in SEQ ID NO:1 or SEQ ID NO: 2, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any embodiments, the target site for DLL4 is a target site having the sequence set forth in SEQ ID NO:1 or SEQ ID NO: 2.

[0011] In some of any embodiments, the target site for DLL4 is a target site having the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 154. In some of any embodiments, the target site for DLL4 has the sequence set forth in SEQ ID NO: 154 or a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence thereof. In some of any embodiments, the target site for DLL4 has the sequence set forth in SEQ ID NO: 154. In some of any embodiments, the target site for DLL4 has the sequence set forth in SEQ ID NO:2 or a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence thereof. In some of any embodiments, the target site for DLL4 has the sequence set forth in SEQ ID NO:2.

[0012] In some of any embodiments, the target site for VCAM1 is in the gene or a regulatory DNA element thereof. In some embodiments, the regulatory DNA element is an enhancer or a promoter of the gene. In some embodiments, the regulatory DNA element is apromoter of the gene. In some of any embodiments, the target site for VCAM1 is within 1000 base pairs of the transcription start site (TSS) of the gene. In some of any embodiments, the target site for VCAM1 is within 20 base pairs, 50 base pairs, 100 base pairs, 200 base pairs, 300 base pairs, 400 base pairs, 500 base pairs, 600 base pairs, or any value between any of the foregoing, of the TSS of the gene.In some of any embodiments, the target site for VCAM1 is within 550 base pairs upstream of the TSS of the gene. In some of any embodiments, the target site for VCAM1 is within the genomic coordinates chrl:100,719,182 to chrl: 100,720,290. In some of any embodiments, the target site for VCAM1 is within the genomic coordinates chrl: 100,566,827 to chrl:100,569,107.

[0013] In some of any embodiments, the target site for VCAM1 is a target site having the sequence set forth in SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any embodiments, the target site for VCAM1 is a target site having the sequence set forth in SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. In some of any embodiments, the target site for VCAM1 has the sequence set forth in SEQ ID NO: 3 or a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence thereof. In some of any embodiments, the target site for VCAM1 has the sequence set forth in SEQ ID NO: 3.

[0014] In some of any embodiments, the DNA-targeting system does not introduce a genetic disruption or a DNA break.In some of any embodiments, the fusion protein of the first DNA- targeting module and / or the second DNA-targeting module comprises a DNA-binding domain selected from: a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or a variant thereof; a zinc finger protein (ZFP); a transcription activator- like effector (TALE); a meganuclease; a homing endonuclease; or an LScel enzyme or a variant thereof, optionally wherein the DNA-binding domain comprises a catalytically inactive variant of any of the foregoing, wherein, when the DNA-binding domain of the fusion protein comprises a Cas protein, the first DNA-targeting module comprises a first guide nucleic acid for targeting the Cas protein to the target site of the DLL4 gene and / or the second DNA-targeting module comprises a second guide nucleic acid for targeting the Cas protein to the target site of the VCAM1 gene. In some of any embodiments, the DNA-binding domain is a zinc finger protein.

[0015] In some of any embodiments, the fusion protein of each of the plurality of DNA- targeting modules is different. In some of any embodiments, the DNA-targeting systemcomprises one fusion protein that is shared by each of the DNA-targeting modules and wherein each DNA-targeting module is characterized by comprising a different guide nucleic acid for targeting the DNA-binding domain to the target site. In some of any embodiments, the first DNA-targeting molecule comprises a first guide nucleic acid for targeting a target site for DLL4 and the second DNA-targeting molecule comprises a second guide nucleic acid for targeting a target site for VCAM1.

[0016] In some of any embodiments, the DNA-binding domain of the fusion protein is a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof. In some of any embodiments, the DNA-binding domain of the first DNA-targeting module and the second DNA-targeting module is a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof, and the first DNA-targeting module comprises at least a first guide nucleic acid for targeting the Cas protein to the target site for DLL4 and the second DNA-targeting module comprises at least a second guide nucleic acid for targeting the Cas protein to the target site for VCAMl.In some of any embodiments, the guide nucleic acid is a guide RNA (gRNA). In some of any embodiments, the Cas protein or variant thereof is a deactivated (dCas) protein.

[0017] Provided herein is a DNA targeting system comprising: (a) a fusion protein comprising a DNA-binding domain comprising a deactivated Cas (dCas) protein and at least one transcriptional activator effector domain; (b) a plurality of gRNAs comprising a first gRNA that targets a target site for DLL4 and a second gRNA that targets a target site for VCAM1. In some embodiments, the DNA-targeting system increases transcription of the DLL4 gene and increases transcription of the VCAM1 gene. In some of any embodiments, the dCas protein lacks nuclease activity .In some of any embodiments, the dCas protein is a dCas9 protein.

[0018] In some of any embodiments, the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein. In some embodiments, the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 88. In some of any embodiments, the dSaCas9 protein comprises the sequence set forth in SEQ ID NO: 89, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any embodiments, the dSaCas9 protein is set forth in SEQ ID NO: 89.

[0019] In some of any embodiments, the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein. In some embodiments, the dSpCas9 protein comprises at least one aminoacid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO: 31. In some of any embodiments, the dSpCas9 protein comprises the sequence set forth in SEQ ID NO: 32, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any embodiments, the dSpCas9 protein is set forth in SEQ ID NO: 32.

[0020] In some of any embodiments, the first gRNAs comprise a gRNA spacer that is complementary to the target site of DLL4. In some of any embodiments, the first gRNA targeting a target site for DLL4 comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 8, 9, or 167-186, or a contiguous portion thereof of at least 14 nt. In some of any embodiments, first gRNA targeting a target site for DLL4 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 174, or a contiguous portion thereof of at least 14 nt. In some of any embodiments, the first gRNA targeting a target site for DLL4 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 174. In some of any embodiments, the first gRNA targeting a target site for DLL4 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9, or a contiguous portion thereof of at least 14 nt. In some of any embodiments, the first gRNA targeting a target site for DLL4 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9. In some of any embodiments, the first gRNA targeting a target site for DLL4 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 9.

[0021] In some of any embodiments, the second gRNA comprises a gRNA spacer that is complementary to the target site of VCAM1. In some of any embodiments, the second gRNA targeting a target site of VCAM1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, or a contiguous portion thereof of at least 14 nt. In some of any embodiments, the second gRNA targeting a target site of VC AMI comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14.In some of any embodiments, the second gRNA targeting a target site for VCAM1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 10.

[0022] In some of any embodiments, the plurality of DNA-targeting modules further comprise one or more additional DNA-targeting modules for increasing transcription of one ormore lymphoid cell differentiation (LCD) genes, wherein each of the one or more additional DNA-targeting modules comprises a fusion protein comprising: (a) a DNA-binding domain that binds to a target site for one of the one or more LCD genes; and (b) at least one transcriptional activator effector domain. In some embodiments, the DNA-binding domain of each of the one or more additional DNA-targeting modules is a deactivated Cas (dCas) protein, and wherein each of the one or more additional DNA-targeting module comprises a guide nucleic acid for targeting the Cas protein to the target site of one of the one or more LCD genes.

[0023] In some of any embodiments, the DNA-targeting system comprises a plurality of DNA-targeting modules comprising the first and / or second DNA-targeting module and the one or more additional DNA-targeting modules, wherein the DNA-targeting system comprises one fusion protein that is shared by each of the plurality of DNA-targeting modules and wherein each DNA-targeting module is characterized by comprising a different guide nucleic acid for targeting the DNA-binding domain to the respective target site, optionally wherein each different guide nucleic acid is a gRNA.

[0024] In some of any embodiments, the DNA-targeting system comprises: (1) a fusion protein comprising (a) a deactivated Cas (dCas) protein; and (b) at least one transcriptional activator effector domain; (2) a guide nucleic acid, optionally a gRNA, for targeting the Cas protein to the target site for DLL4; (3) a guide nucleic acid, optionally a gRNA, for targeting the Cas protein to the target site for VCAM1; and (4) one or more additional guide nucleic acids, optionally at least one or more additional gRNA, for targeting the Cas protein to the target site of each of the one or more LCD genes.

[0025] In some of any embodiments, the DNA-targeting system comprises (1) a fusion protein comprising (a) a deactivated Cas (dCas) protein; and (b) at least one transcriptional activator effector domain; (2) a gRNA targeting a target site for DLL4; (3) a gRNA targeting a target site for VCAM1; and (4) one or more gRNA targeting a target site of the one or more LCD genes, optionally wherein each of the one or more gRNA targets a target site of one of the LCD genes. In some of any embodiments, the DNA-targeting system comprises 3 to 10 different gRNAs, optionally 3 to 6 different gRNAs.

[0026] In some of any embodiments, the one or more lymphoid cell differentiation (LCD) genes is selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL11B. In some of any embodiments, the one or more additional DNA-targeting modules is one additional DNA-targeting module, wherein theone additional DNA-targeting module targets a lymphoid cell differentiation (LCD) gene selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL11B. In some of any embodiments, the one or more additional DNA-targeting modules target two or more LCD genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL1 IB. In some of any embodiments, the LCD genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

[0027] In some of any embodiments, at least one LCD gene is RUNX3. In some of any embodiments, the DNA-targeting system comprises:(l) a fusion protein comprising (a) a deactivated Cas (dCas) protein; and (b) at least one transcriptional activator effector domain; (2) a gRNA targeting a target site for DLL4; (3) a gRNA targeting a target site for VCAM1; and (4) a gRNA targeting a target site for RUNX3.

[0028] In some of any embodiments, at least one LCD gene is RUNX1. In some of any embodiments, at least one LCD gene is LEFl.In some of any embodiments, at least one LCD gene is MYB. In some of any embodiments, at least one LCD gene is TCF7. In some of any embodiments, at least one LCD gene is BCL1 IB. In some of any embodiments, the one or more LCD genes are TCF7, BCL1 IB, and MYB. In some of any embodiments, the LCD genes are TCF7, MYB, and RUNX3.

[0029] In some of any embodiments, the target site for each of the one or more LCD genes is in the gene or a regulatory DNA element thereof. In some of any embodiments, the regulatory DNA element is an enhancer or a promoter of the gene. In some of any embodiments, the regulatory DNA element is a promoter of the gene. In some of any embodiments, the target site for each of the one or more LCD genes is within 1000 base pairs of the transcription start site (TSS) of the gene. In some of any embodiments, the target for each of the one or more LCD genes is within 20 base pairs, 50 base pairs, 100 base pairs, 200 base pairs, 300 base pairs, 500 base pairs, 600 base pairs, or any value between any of the foregoing, of the TSS of the gene. In some of any embodiments, the target site for each of the one or more LCD genes is within 550 base pairs of the TSS of the gene.

[0030] In some of any embodiments, the target site for RUNX3 has the sequence forth in SEQ ID NO: 46 or SEQ ID NO: 47, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any embodiments, the target site for RUNX3 has the sequence forth in SEQ ID NO: 46. In some of any embodiments, thegRNA targeting a target site for RUNX3 comprises a gRNA spacer sequence comprising the sequence forth in SEQ ID NO: 60 or SEQ ID NO: 61, or a contiguous portion thereof of at least 14 nucleotides (nt). In some of any embodiments, the gRNA spacer sequence comprises the sequence forth in SEQ ID NO: 60.

[0031] In some of any embodiments, the dCas protein is a dCas9 protein. In some of any embodiments, the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein. In some of any embodiments, the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein. In some of any embodiments, the guide nucleic acid is a gRNA. In some of any embodiments, each gRNA independently comprises a spacer sequence between 14 nt and 24 nt, or between 16 nt and 22 nt in length. In some of any embodiments, each gRNA independently comprises a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length. In some of any embodiments, each gRNA independently further comprises a scaffold sequence set forth in SEQ ID NO: 83. In some of any embodiments, each gRNA independently further comprises a scaffold sequence set forth in SEQ ID NO: 23. In some of any embodiments, each gRNA further comprises 2’ MeO modified bases and / or phosphorothiate backbone modifications.

[0032] In some of any embodiments, the at least one transcriptional activator effector domain is selected from the group consisting of: a VP64 domain, a p65 activation domain, a p300 domain, an Rta domain, a CBP domain, a VPR domain, a VPH domain, an HSF1 domain, a TET protein domain, optionally wherein the TET protein is TET1, a SunTag domain, or a domain, portion, variant, or truncation of any of the foregoing. In some of any embodiments, the at least one transcriptional activator effector domain comprises at least one VP 16 domain, and / or a VP16 tetramer (“VP64”) or a variant thereof. In some of any embodiments, the at least one transcriptional activator effector domain comprises a VP64 domain or a variant or portion thereof that exhibits transcriptional activation activity. In some of any embodiments, the at least one transcriptional activator effector domain is VP64. In some embodiments, the VP64 is positioned N-terminal and / or C-terminal to the DNA-binding domain. In some of any embodiments, the at least one transcriptional activator effector domain comprises two copies of VP64.

[0033] In some of any embodiments, the at least one transcriptional activator effector domain comprises the amino acid sequence set forth in SEQ ID NO: 28, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 28. In some of any embodiments, the at least onetranscriptional activator effector domain comprises the amino acid sequence set forth in SEQ ID NO: 30, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 30. In some of any embodiments, the fusion protein comprises the sequence set forth in SEQ ID NO: 26, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0034] In some of any embodiments, increasing transcription of DLL4 and VCAM1, and optionally increasing transcription of the one or more LCD genes, promotes differentiation of a hematopoietic progenitor cell (HPC) to a lymphoid progenitor.In some of any embodiments, transient delivery of the DNA-targeting system to a hematopoietic progenitor cell (HPC) promotes differentiation to a lymphoid progenitor. In some embodiments, the lymphoid progenitor is an induced common lymphoid progenitor (iCLP).In some of any embodiments, transient delivery of the DNA-targeting system to an HPC promotes differentiation to a CD5+ cell. In some of any embodiments, transient delivery of the DNA-targeting system to an HPC promotes differentiation to a CD7+ cell. In some of any embodiments, transient delivery of the DNA-targeting system to an HPC promotes differentiation to a CD5+ / CD7+ cell. In some of any embodiments, the HPC is an induced hematopoietic progenitor cell (iHPC).

[0035] Provided herein is a gRNA that targets a target site for DLL4 target site for DLL4 comprises the sequence set forth in any one of SEQ ID NOs:l-2 or 147-166, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any embodiments, the target site for DLL4 comprises the sequence set forth SEQ ID NO: 154, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any embodiments, the target site for DLL4 comprises the sequence set forth SEQ ID NO: 154. In some of any embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 8,9 and 167-186, or a contiguous portion thereof of at least 14 nt. In some of any embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 174, or a contiguous portion thereof of at least 14 nt.

[0036] Provided herein is a gRNA that targets a target site for DLL4, wherein the target site for DLL4 comprises the sequence set forth in any one of SEQ ID NOS: 1-2 a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site for DLL4 comprises the sequence set forth in any one of SEQID NOS: 1-2. In some of any embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9, or a contiguous portion thereof of at least 14 nt. In some embodiments, the gRNA spacer sequence comprises the sequence set forth in SEQ ID NO: 8 or SEQ ID NO:9.

[0037] Provided herein is a gRNA that targets a target site for VCAM1, wherein the target site for VCAM comprises the sequence set forth in any one of SEQ ID NOS:3-7 a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site for VCAM comprises the sequence set forth in any one of SEQ ID NOS:3-7. In some of any embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, or a contiguous portion thereof of at least 14 nt. In some embodiments, the gRNA spacer sequence comprises the sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14.

[0038] In some of any embodiments, the spacer sequence is between 14 nt and 24 nt, or between 16 nt and 22 nt in length. In some of any embodiments, the spacer sequence is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length. In some of any embodiments, the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 23. In some of any embodiments, the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 83. In some of any embodiments, the gRNA further comprises 2’ MeO modified bases and / or phosphorothiate backbone modifications.

[0039] Also provided herein is a combination comprising two or more gRNAs of some of any embodiments. In some embodiments, at least one gRNA targets a target site in DLL4 and at least one gRNA targets a target site in VCAM1. Also provided herein is a combination of gRNAs comprising a first gRNA that targets a target site for DLL4 and a second gRNA that targets a target site for VCAM1. In some embodiments, the target site for DLL4 is within DLL4 or a regulatory element thereof. In some of any embodiments, the target site for VCAM1 is within VCAM1 or a regulatory element thereof. In some of any embodiments, the regulatory DNA element is an enhancer or a promoter. In some of any embodiments, the regulatory DNA element is a promoter. In some of any embodiments, the target site for DLL4 is within an enhancer or promoter.

[0040] In some of any embodiments, the target site for DLL4 is within 1000 base pairs of the transcription start site (TSS) of DLL4. In some of any embodiments, the target site for DLL4is within 20 base pairs, 50 base pairs, 100 base pairs, 200 base pairs, 300 base pairs, 400 base pairs, 500 base pairs, 600 base pairs, or any value between any of the foregoing, of each TSS. In some of any embodiments, the target site for DLL4 and the target site for VCAM1 is with 550 base pairs upstream of each TSS.

[0041] In some of any embodiments, the target site for DLL4 has a sequence set forth in any one of SEQ ID NOs:l, 2 or 147-166, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any embodiments, the target site for DLL4 has a sequence set forth in SEQ ID NO: 154 or a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence thereof. In some of any embodiments, the target site for DLL4 has a sequence set forth in SEQ ID NO: 154. In some of any embodiments, the target site for DLL4 has a sequence set forth in SEQ ID NO:1 or SEQ ID NO: 2, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any embodiments, the target site for DLL4 has a sequence set forth in SEQ ID NO:1 or SEQ ID NO: 2.In some of any embodiments, the target site for DLL4 has a sequence set forth in SEQ ID NO:2 or a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence thereof. In some of any embodiments, the target site for DLL4 has a sequence set forth in SEQ ID NO:2.

[0042] In some of any embodiments, the first gRNA comprises a gRNA spacer that is complementary to the target site of DLL4. In some of any embodiments, the first gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 174, or a contiguous portion thereof of at least 14 nt. In some of any embodiments, the first gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 174. In some of any embodiments, the first gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9, or a contiguous portion thereof of at least 14 nt. In some of any embodiments, the first gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9. In some of any embodiments, the first gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 9.

[0043] In some of any embodiments, the target site for VCAM1 is within an enhancer or promoter. In some of any embodiments, the target site for VC AMI is within 1000 base pairs of the transcription start site (TSS) of VCAM1. In some of any embodiments, the target site forVCAM1 is within 20 base pairs, 50 base pairs, 100 base pairs, 200 base pairs, 300 base pairs, 400 base pairs, 500 base pairs, 600 base pairs, or any value between any of the foregoing, of the TSS. In some of any embodiments, the target site for VCAM1 is with 550 base pairs upstream of each TSS.

[0044] In some of any embodiments, the target site for VCAM1 has a sequence set forth in SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any embodiments, the target site for VCAM1 has a sequence set forth in SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. In some of any embodiments, the target site for VCAM1 has a sequence set forth in SEQ ID NO:3 or a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence thereof. In some of any embodiments, the target site for VCAM1 has a sequence set forth in SEQ ID NO:3.

[0045] In some of any embodiments, the second gRNA comprises a gRNA spacer that is complementary to the target site of VCAM1. In some of any embodiments, the second gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or a contiguous portion thereof of at least 14 nt. In some of any embodiments, the second gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14. In some of any embodiments, the second gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 10.

[0046] In some of any embodiments, the first gRNA and the second gRNA independently comprise a spacer sequence between 14 nt and 24 nt, or between 16 nt and 22 nt in length. In some of any embodiments, the first gRNA and the second gRNA independently comprise a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length. In some of any embodiments, the first gRNA and the second gRNA each further comprise a scaffold sequence set forth in SEQ ID NO: 23. In some embodiments, the first gRNA and the second gRNA each further comprise a scaffold sequence set forth in SEQ ID NO: 83. In some of any embodiments, the first gRNA and / or the second gRNA independently further comprise 2’ MeO modified bases and / or phosphorothiate backbone modifications.

[0047] In some of any embodiments, the combination of gRNAs further comprises one or more additional gRNAs, wherein the one or more additional gRNAs target a target site of one ormore lymphoid cell differentiation (LCD) genes, wherein the one or more LCD genes is selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL1 IB. In some embodiments, the one or more additional gRNAs is one additional gRNA, wherein the one additional gRNA targets a target site of an LCD gene selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL11B. In some of any embodiments, the additional gRNA targets a target site of RUNX3. In some of any embodiments, the target site for RUNX3 has the sequence forth in SEQ ID NO: 46 or SEQ ID NO: 47, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any embodiments, the target site for RUNX3 has the sequence forth in SEQ ID NO: 46. In some embodiments, the one or more additional gRNAs are two gRNAs, wherein the two additional gRNAs target a target sites of two or more LCD genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL1 IB, or any combination thereof.

[0048] In some of any embodiments, the LCD genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some of any embodiments, at least one LCD gene is RUNX3. In some of any embodiments, at least one LCD gene is RUNX1. In some of any embodiments, at least one LCD gene is LEF1. In some of any embodiments, at least one LCD gene is MYB. In some of any embodiments, at least one LCD gene is TCF7. In some of any embodiments, at least one LCD gene is BCL1 IB. In some of any embodiments, the one or more LCD genes are TCF7, BCL1 IB, and MYB. In some of any embodiments, wherein the LCD genes are TCF7, MYB, and RUNX3.

[0049] Also provided herein is a Cas-guide RNA (gRNA) combination comprising: (a) a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof fused to a transcriptional activation domain; and (b) a combination of gRNAs of some of any embodiments. Also provided herein is a Cas-guide RNA (gRNA) combination comprising: (a) a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof fused to a transcriptional activation domain; and (b) one or more gRNAs of some of any embodiments.

[0050] In some of any embodiments, the Cas protein or variant thereof is a deactivated (dCas) protein. In some embodiments, the dCas protein lacks nuclease activity. In some of any embodiments, the dCas protein is a dCas9 protein. In some embodiments, the dCas9 protein is aStaphylococcus aureus dCas9 (dSaCas9) protein. In some embodiments, the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 88. In some of any embodiments, the dSaCas9 protein comprises the sequence set forth in SEQ ID NO: 89, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any embodiments, the dSaCas9 protein is set forth in SEQ ID NO: 89.

[0051] In some of any embodiments, the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein. In some embodiments, the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO: 31. In some of any embodiments, the dSpCas9 protein comprises the sequence set forth in SEQ ID NO: 32, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any embodiments, the dSpCas9 protein is set forth in SEQ ID NO: 32.

[0052] In some of any embodiments, the at least one transcriptional activator effector domain comprises at least one VP 16 domain, and / or a VP 16 tetramer (“VP64”) or a variant thereof. In some of any embodiments, the at least one transcriptional activator effector domain comprises a VP64 domain or a variant or portion thereof that exhibits transcriptional activation activity. In some of any embodiments, the at least one transcriptional activator effector domain is VP64. In some of any embodiments, the VP64 is positioned N-terminal and / or C-terminal to the DNA-binding domain. In some of any embodiments, the at least one transcriptional activator effector domain comprises two copies of VP64. In some of any embodiments, the at least one transcriptional activator effector domain comprises the amino acid sequence set forth in SEQ ID NO: 28, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 28. In some of any embodiments, the at least one transcriptional activator effector domain comprises the amino acid sequence set forth in SEQ ID NO: 30, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 30. In some of any embodiments, the fusion protein comprises the sequence set forth in SEQ ID NO: 26, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any embodiments, the polynucleotide encoding the fusion protein is an mRNA.

[0053] Also provided herein is a polynucleotide encoding the DNA-targeting system of some of any embodiments. Also provided herein is a polynucleotide encoding at least one DNA- targeting module of the DNA-targeting system of some of any embodiments. Also provided herein is a polynucleotide encoding the gRNA of some of any embodiments. Also provided herein is a polynucleotide encoding the combination of gRNAs of some of any embodiments. Also provided herein is a polynucleotide encoding the Cas-gRNA combination of some of any embodiments.

[0054] Also provided herein is a plurality of polynucleotides encoding any DNA-targeting system, any combination of gRNAs, any Cas-gRNA combination, or any portion or a component of any of the foregoing of some of any embodiments. Also provided herein is a plurality of polynucleotides encoding any DNA-targeting system, any combination of gRNAs, any Cas-gRNA combination, or any portion or a component of any of the foregoing of some of any embodiments.

[0055] Also provided herein is a plurality of polynucleotides encoding the epigenetic- modifying DNA-targeting system of some of any embodiments. Also provided herein is a plurality of polynucleotides encoding at least one DNA-targeting module of the epigenetic- modifying DNA-targeting system of some of any embodiments. Also provided herein is a plurality of polynucleotides encoding the fusion protein and the at least first gRNA and second gRNA of the epigenetic-modifying DNA-targeting system of some of any embodiments. Also provided herein is a vector comprising the polynucleotide of some of any embodiments. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a lipid nanoparticle.

[0056] Also provided herein is a pharmaceutical composition comprising the DNA-targeting system of some of any embodiments, the Cas-gRNA combination of some of any embodiments, the polynucleotide of some of any embodiments, or the vector of some of any embodiments. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0057] Also provided herein is a method of differentiating a population of hematopoietic progenitor cells (HPCs) to a differentiated population of cells, the method comprising introducing the DNA-targeting system of some of any embodiments, the Cas-gRNA combination of some of any embodiments, the polynucleotide of some of any embodiments, thevector of some of any embodiments, or a combination thereof, into a population of HPCs, and culturing the HPCs under conditions for their differentiation.

[0058] Also provided herein is a method of differentiating a population of hematopoietic progenitor cells (HPCs) to a differentiated population of cells, the method comprising introducing the pharmaceutical composition of some of any embodiments into a population of hematopoietic progenitor cells (HPCs) and culturing the HPCs under conditions for their differentiation. In some embodiments, the population of HPCs are induced hematopoietic progenitor cells (iHPCs). In some embodiments, the population of HPCs are primary hematopoietic progenitor cells.

[0059] In some of any embodiments, the HPCs comprise cells engineered with a recombinant receptor, optionally a chimeric antigen receptor. In some of any embodiments, the differentiated cells comprise cells that express a recombinant receptor, optionally a chimeric antigen receptor. Also provided herein is a population of differentiated cells produced by the method of some of any embodiments.

[0060] In some of any embodiments, cells of the differentiated population of cells are lymphoid progenitor cells. In some embodiments, the lymphoid progenitor cells are induced common lymphoid progenitor cells (iCLPs). In some of any embodiments, the lymphoid progenitor cells are CD5+ cells. In some of any embodiments, the lymphoid progenitor cells have increased CD5 expression relative to the population of HPCs. In some of any embodiments, the lymphoid progenitor cells are CD7+ cells. In some of any embodiments, the lymphoid progenitor cells have increased CD7 expression relative to the population of HPCs. In some of any embodiments, the lymphoid progenitor cells are CD5+CD7+ cells. In some of any embodiments, the lymphoid progenitor cells have increased CD5 and CD7 expression relative to the population of HPCs. In some of any embodiments, the lymphoid progenitor cells are CD45RA+ cells. In some of any embodiments, the lymphoid progenitor cells have increased CD45RA+ expression relative to the population of HPCs. In some of any embodiments, the lymphoid progenitor cells are CD117-mid cells. In some of any embodiments, the lymphoid progenitor cells have increased CD117 expression relative to the population of HPCs. In some of any embodiments, the lymphoid progenitor cells are CD34- cells. In some of any embodiments, the lymphoid progenitor cells have decreased CD34 expression relative to the population of HPCs. In some of any embodiments, the lymphoid progenitor cells are CD 123- cells. In some of any embodiments, the lymphoid progenitor cells have decreased CD 123expression relative to a population of monocytes. In some of any embodiments, the lymphoid progenitor cells are CD14- cells. In some of any embodiments, the lymphoid progenitor cells have decreased CD14 expression relative to a population of monocytes. In some of any embodiments, the lymphoid progenitor cells are CD56- cells. In some of any embodiments, the lymphoid progenitor cells have decreased CD56 expression relative to a population of lymphocytes.In some of any embodiments, the lymphoid progenitor cells express a recombinant receptor, optionally a chimeric antigen receptor. In some of any embodiments, cells of the differentiated population of cells are lymphoid cells (LCs).Also provided herein is a population of lymphoid progenitor cells produced by the method of some of any embodiments.

[0061] Also provided herein is a method of generating lymphoid cells (LCs), the method comprising culturing the population of lymphoid progenitor cells produced by the method of some of any embodiments, or the population of lymphoid progenitor cells of some of any embodiments under conditions to differentiate cells of the population to lymphoid cells (LCs) to produce a population comprising LCs. In some of any embodiments, the differentiated population of cells are lymphoid cells (LCs).

[0062] In some of any embodiments, the LCs are induced Natural Killer (iNK) cells. In some of any embodiments, the LCs are induced Natural Killer (iNK) cells. In some embodiments, the iNK cells are CD56+CD3- cells. In some of any embodiments, the iNK cells are further characterized by one or more of the following: DNAM1+, NKG2D+, NKP30+ and / or CD16+.

[0063] Provided herein is a method of generating induced T (iT) cell progenitors, the method comprising contacting the population of lymphoid progenitor cells of some of any embodiments with a coating comprising exogenous DLL4 ligand. In some of any embodiments, the coating further comprises retronectin. In some of any embodiments, the iT cell progenitors are double positive CD4+ / CD8+ cells. Provided herein is a population of iT cell progenitors produced by the method of some of any embodiments. In some of any embodiments, the LCs are induced T (iT) cells.

[0064] Provided herein is a method of generating iT cells, the method comprising culturing the population of iT cell progenitors produced by the method or the population of lymphoid progenitor cells of some of any embodiments under conditions to differentiate cells of the population to iT cell progenitors to produce a population comprising iT cells.

[0065] In some embodiments, the iT cells are characterized by one or more of the following: CD2+, CD3+, CD4+, and / or CD8b+. In some of any embodiments, the iT cells are CD4+ cells. In some of any embodiments, the iT cells are CD8b+ cells. In some of any embodiments, the iT cells are CD8+ single positive cells. In some of any embodiments, the iT cells are CD8b+ / CD8a+. In some of any embodiments, the LCs express a recombinant receptor, optionally a chimeric antigen receptor.

[0066] Also provided herein is a population of lymphoid cells produced by the method of some of any embodiments. In some of any embodiments, the method is carried out in vitro or ex vivo. In some of any embodiments, the HPCs are human HPCs. In some of any embodiments, the introducing is by transient delivery into the population of HPCs. In some embodiments, the transient delivery comprises electroporation, transfection, or transduction.

[0067] In some of any embodiments, the introducing increases transcription of DLL4 and VCAMl.In some of any embodiments, the introducing also increases transcription of the one or more lymphoid cell differentiation (LCD) genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL11B.

[0068] Also provided herein is a method of treating a disease or condition in a subject, the method comprising administering to the subject the population of the population of differentiated cells of some of any embodiments, the population of lymphoid progenitors of some of any embodiments, or the population of lymphoid cells of some of any embodiments.Brief Description of the Drawings

[0069] FIG. 1 shows results from qRT-PCR to assess expression of DLL4 and VCAM1 mRNA in hematopoietic progenitor cells (HPCs) treated with LNPs comprising dSpCas9- 2xVP64 and gRNAs targeting each gene. Expression is shown as fold change relative to the non-targeting gRNA control (NTC) and normalized to GADPH.

[0070] FIGs. 2A-2B show surface marker expression of DLL4 and VCAM1 following LNP-mediated delivery of dSpCas9-2xVP64 and gRNAs targeting each gene or a non-targeting (NT) negative control. FIG. 2A shows representative flow cytometry plots of DLL4 and VCAM1 surface marker expression. FIG. 2B shows a quantification of DLL4 and VCAM1 surface marker expression up to 10 days following LNP-mediated delivery of dSpCas9-2xVP64 and gRNAs targeting each gene, non-targeting negative control cells (NT CTRL), or positive control cells differentiated in cultures with lymphoid coating material (POST CTRL_LCM).

[0071] FIG. 3 shows the protocol for differentiation of induced hematopoietic progenitor cells (iHPCs) to lymphoid progenitor cells (induced common lymphoid progenitors; iCLPs) using LNP-mediated delivery of dSpCas9-2xVP64 and gRNAs targeting DLL4 and VCAM1 at DO and D4.

[0072] FIGs. 4A-4F show changes in surface marker expression following treatment of hematopoietic progenitor cells with LNP-mediated delivery of dSpCas9-2xVP64 and gRNAs targeting DLL4 and VCAM1 (DLL4+VCAM1), positive control differentiation of lymphoid progenitors through culture with lymphoid coating material (LCM-positive control), and nontargeting gRNA negative control cells (NT Control). FIG. 4A shows representative flow cytometry plots for CD5 and CD7 expression between all three populations, while FIG. 4B shows quantification for percentage of CD5+CD7+ cells resulting from each treatment condition. FIG. 4C shows representative flow cytometry plots for CD45RA expression between all three populations. Results are shown for cells gated using the following gating strategy: Single cells >Live> CD45RA. FIG. 4D shows representative flow cytometry plots for CD117 and CD56 expression between all three populations. Results are shown for cells gated using the following gating strategy: Single cells>Live> CD45+>CD5+CD7+> CD56x Ckit mid. FIG. 4E shows representative flow cytometry plots for CD 14 and CD 123 expression between all three populations. Results are shown for cells gated using the following gating strategy: Single cells>Live> CD123xCD14. FIG. 4F shows representative flow cytometry plots for CD34 expression between all three populations. Results are shown for cells gated using the following gating strategy: Single cells>Live> CD34.

[0073] FIG. 5 shows the fold-change expansion of iCLPs over time following LNP- mediated delivery of dSpCas9-2xVP64 and gRNAs targeting DLL4 and VCAM1 (DLL4+VCAM1), positive control differentiation of lymphoid progenitors through culture with lymphoid coating material (POS), and non-targeting gRNA negative control cells (NT).

[0074] FIG. 6 shows the protocol for differentiation of induced Natural Killer (iNK) cells from iHPCs using LNP-mediated delivery of dSpCas9-2xVP64 and gRNAs targeting DLL4 and VC AMI with or without DLL4 and retronectin (DLL4+Ret) culture coating.

[0075] FIGS. 7A-B shows quantification of surface marker expression after differentiation of iNKs with or without DLL4+Ret culture coating. iNKs were differentiated from iCLPs generated by LNP-mediated delivery of dSpCas9-2xVP64 and gRNAs targeting DLL4 and VCAM1 or by lymphoid differentiation coating material (LCM) -mediated differentiation (POSCTRL). FIG. 7A shows a quantification of CD45 and CD56 expression on the left and a quantification of CD 16 expression on the right. FIG. 7B shows a quantification of (from top to bottom) NKG2D, NKP30, and DNAM expression.

[0076] FIGs. 8A-8D show the results of a tumor-killing assay wherein cells were cocultured with HER2+ NSCLC tumor cell line (Hl 975) in a first round of co-culture and a second round of co-culture with or without DLL4+Ret coating iNKs generated from iCLPs differentiation through LNP-mediated delivery of dSpCas9-2xVP64 and gRNAs targeting DLL4 and VCAM1 (DLL4+VCAM1). Negative controls included T cells without CAR specific to HER2 (T cell control) and the HER2+ NSCLC cell line alone (target alone). Positive controls included anti-HER2-CAR-T cells (CAR T cell control) and iNKs derived from LCM- differentiated lymphoid progenitor cells (POS CTRL). FIG. 8A shows the results for the first round of co-culture with the DLL4+Ret coating. FIG. 8B shows the results for the first round of co-culture with no coating. FIG. 8C shows the results for the second round of co-culture with the DLL4+Ret coating. FIG. 8D shows the results for the second round of co-culture with no coating.

[0077] FIGs. 9A-9C show results for a transient CRISPR activation screen. FIG. 9A shows an exemplary workflow for a transient CRISPR activation screen. Hematopoietic Progenitor Cells (HPCs) are transduced with a lentiviral gRNA library and transfected with lipid nanoparticles (LNPs) for delivery of an epi-editor, such as a dCas fused to a transcriptional activator. Cells are then screened for the desired cell surface marker phenotype using flow cytometry. FIG. 9B shows an exemplary flow cytometry plot showing expression of CD45 in HPCs delivered with the gRNA library and epi-editor, with a box indicating the CD45high populations to be sorted for further analysis. FIG. 9C shows an exemplary plot from the CRISPR activation screen for gRNAs and genes that modulate CD45 expression. Dots represent individual gRNAs. gRNAs on the right are those that target genes whose activation results in increased CD45 expression. X-axis represents log2 fold change of gRNA abundance in CD45high sorted cells versus unsorted cells. Y-axis represents significance (-loglO adjusted p- value).

[0078] FIGS. 10A-10 B show results from RT-qPCR to assess transcription factor expression following targeted activation using a DNA-targeting system. Hematopoietic progenitor cells (HPCs) were transfected on Day 0 with LNPs for delivery of mRNA encoding dSpCas9-2xVP64 and a single gRNA targeting one of the genes or a combination of three guideRNAs, each targeting a different gene. Expression is shown as fold change relative to a nontargeting (NT) gRNA control. Results are shown for Day 2 post-transfection. FIG. 10A shows results for MYB, RUNX1, HEY1, RUNX3, and SPI expression. FIG. 10B shows results for IL7Ra, TBX21, LEF1, or CBFB expression.

[0079] FIGs. 11A-11C show results of ectopic expression of LCD genes in HPCs. FIG. 11A shows flow cytometry plots for assessing expression of CD45RA and CD34. HPCs were transfected on Day 0 with LNPs for delivery of mRNA encoding dSpCas9-2xVP64 with a nontargeting (NT) gRNA or a gRNA targeting HEY1, TBX21, SPI1, or RUNX1. Results are shown for Day 4 post-transfection with a box indicating the CD45RA+CD34- population. FIG. 11B shows flow cytometry plots for assessing expression of CD7 and CD 14. HPCs were transfected on Day 0 with LNPs for delivery of mRNA encoding dSpCas9-2xVP64 with a non-targeting (NT) gRNA or a gRNA targeting HEY1, TBX21, SPI1, or RUNX1. Results are shown for Day 4 post-transfection. FIG. 11C shows flow cytometry plots for assessing expression of CD7 and CD5. HPCs were transfected on Day 0 and Day 4 with LNPs for delivery of mRNA encoding dSpCas9-2xVP64 with a non-targeting (NT) gRNA or a gRNA targeting LEF1 or MYB. Results are shown for Day 7 post-transfection.

[0080] FIGs. 12A-12J show flow cytometry plots for assessing expression of cell surface markers of HPCs transfected on Day 0 and Day 4 with LNPs for delivery of mRNA encoding dSpCas9-2xVP64 and multiplexed gRNAs targeting different combinations of three genes. Results are shown for Day 7 post-transfection. FIG. 12A shows expression of CD45Ra and CD34, CD 14 and IL3Ra, CD5 and CD7, and ckit and CD56 for positive control cells cultured in wells coated with Lymphoid Coating Material (LCM). FIG. 12B shows expression of CD45Ra and CD34, CD 14 and IL3Ra, CD5 and CD7, and ckit and CD56 for negative control cells transfected with a non-targeting (NT) gRNA. FIG. 12C shows expression of CD45Ra and CD34, CD 14 and IL3Ra, CD5 and CD7, and ckit and CD56 for cells transfected with gRNAs targeting RUNX3, SPI1, and IL7Ra. FIG. 12D shows expression of CD45Ra and CD34, CD 14 and IL3Ra, CD5 and CD7, and ckit and CD56 for cells transfected with gRNAs targeting RUNX1, HEY1, and TBX21. FIG. 12E shows expression of CD45Ra and CD34, CD14 and IL3Ra, CD5 and CD7, and ckit and CD56 for cells transfected with gRNAs targeting HEY1, IL7Ra, andTBX21. FIG. 12F shows expression of CD45Ra and CD34, CD14 and IL3Ra, CD5 and CD7, and ckit and CD56 for cells transfected with gRNAs targeting MYB, IL7Ra, and LEF1. FIG. 12G shows expression of CD45Ra and CD34, CD 14 and IL3Ra, CD5 and CD7,and ckit and CD56 for cells transfected with gRNAs targeting RUNX3, IL7a, and MYB. FIG. 12H shows expression of CD45Ra and CD34, CD 14 and IL3Ra, CD5 and CD7, and ckit and CD56 for cells transfected with gRNAs targeting RUNX3, IL7a, and RUNX1. FIG. 121 shows expression of CD45Ra and CD34, CD 14 and IL3Ra, CD5 and CD7, and ckit and CD56 for cells transfected with gRNAs targeting RUNX3, IL7a, and TBX21. FIG. 12J shows expression of CD45Ra and CD34, CD 14 and IL3Ra, CD5 and CD7, and ckit and CD56 for cells transfected with gRNAs targeting RUNX3, IL7a, and LEF1.

[0081] FIGs. 13A-13C show plots quantifying cell surface marker expression, as assessed by flow cytometry, for cells derived from HPCs. Cells were transfected on Day 0 and Day 4 with LNPs for delivery of mRNA encoding dSpCas9-2xVP64 and multiplexed gRNAs targeting the combinations of genes summarized in Table E6 and subsequently cultured in Natural Killer (NK) cell induction media. HPCs cultured with Lymphoid Coating Material (LCM) prior to culturing in NK cell induction media served a positive control (POS). Cells were sequentially gated for live cells, CD45+EGFRt+, CD56+, and CD16+, DNAM-1+ or NKG2D+. Results are shown for Day 14 post-transfection as percent of parent gate. FIG. 13A shows percentage of CD45+EGFRt+, CD56+, CD16+, DNAM-1+ and NKG2D+ cells, as assessed by flow cytometry, for cells transfected with gRNA combinations that targeted RUNX3, IL7Ra, and TBX21 on Day 0 (Group 1). FIG. 13B shows percentage of CD45+EGFRt+, CD56+, CD16+, DNAM-1+ and NKG2D+ cells, as assessed by flow cytometry, for cells transfected with gRNA combinations that targeted RUNX3, IL7Ra, and CBFB on Day 0 (Group 2). FIG. 13C shows percentage of CD45+EGFRt+, CD56+, CD16+, DNAM-1+ and NKG2D+ cells, as assessed by flow cytometry, for cells transfected with gRNA combinations that targeted RUNX3 and IL7Ra, on Day 0 (Group 3).

[0082] FIGs. 14A-14B show the cytotoxicity of iNK cells derived from ectopic expression of LCD genes compared to the cytotoxicity of positive and negative control cells. iNK cells were derived from HPCs transfected on Day 0 and Day 4 with LNPs for delivery of mRNA encoding dSpCas9-2xVP64 and multiplexed gRNAs targeting the combinations of genes summarized in Table E6 and subsequently cultured in Natural Killer (NK) cell induction media. Results are shown for cells transfected with gRNA combinations that targeted RUNX3 and IL7Ra, on Day 0 (combinations 3-1 through 3-6). HPCs cultured with Lymphoid Coating Material (LCM) prior to culturing in NK cell induction media served as a positive control (POS). The Hl 975 tumor cell line was cultured alone (target alone) as a negative control. FIG.14A shows exemplary data for a cytotoxicity co-culture assay using H1975 tumor cells and induced Natural Killer (iNK) cells. FIG. 14B shows a zoomed-in version of FIG. 14A to highlight the gene combinations that resulted in robust tumor killing capacity similar to the positive control cells.

[0083] FIGs. 15A-15B show differentiation of mature lymphocytes (z.e. induced T cells; “iT cells”) using ectopic expression of target genes. FIG. 15A shows the complete protocol for differentiating iLymphocytes from hematopoietic progenitor cells. FIG. 15B shows representative flow cytometric analysis of surface marker expression of mature lymphocyte markers CD8b and CD4 in iT cells differentiated with LNP-mediated delivery of dSpCas9- 2xVP64 and gRNAs targeting DLL4 and VCAM1 alone (DLL4+VCAM1), gRNAs targeting DLL4 and VCAM1 alongside the LCD gene RUNX1 (DLL4+VCAM1+RUNX1), and positive control cells differentiated through Kaufmann VO lymphocyte differentiation protocol (VO Positive Control).

[0084] FIGs. 16A-16D show functional analysis of mature lymphocytes (i.e., induced NK cells; “iNK cells”) differentiated using ectopic expression of target genes. FIG. 16A shows the complete protocol for differentiating iNK cells from iHPCs. FIG. 16B shows proliferation capacity after HER2-Fc antigen stimulation of iNK cells differentiated from LNP-mediated delivery of dSpCas9-2xVP64 and gRNAs targeting DLL4 and VCAM1 alone (DLL4+VCAM1), gRNAs targeting DLL4 and VCAM1 alongside the LCD gene RUNX3 (DLL4+VCAM1+RUNX3), iNK cells differentiated from lymphoid progenitors generated from DLL4-coated cultures (DLL4 coating +) and positive control anti-HER2 CAR-T cells. FIG. 16C shows the results of a serial tumor cell-killing co-culture assay with mature lymphocytes and HER2+ NSCLC tumor cell line (Hl 975). iNK cells were differentiated through LNP- mediated delivery of dSpCas9-2xVP64 and gRNAs targeting DLL4 and VCAM1 alone (DLL4+VCAM1), gRNAs targeting DLL4 and VCAM1 alongside the LCD gene RUNX1 (DV+RUNX1), gRNAs targeting DLL4 and VC AMI alongside the LCD gene RUNX3 (DV+RUNX3), gRNAs targeting DLL4 and VCAM1 alongside the LCD gene LEF1 (DV+LEF1), and Kaufmann VO lymphocyte differentiation protocol (POS CTRL). Other controls include T cells without CAR specific to HER2 (T cell control), HER2+ NSCLC cell line alone (target alone), and anti-HER2-CAR-T cells (CAR T cell control). FIG. 16D shows live cell counts of each lymphocyte population following the first round of the serial tumor cellkilling co-culture assay.

[0085] FIGS. 17A-17B show flow cytometry plots for assessing cell surface expression of IL7Ra and VCAM1 in iHPCs transfected with various doses of LNPs for delivery of a multiplex DNA-targeting system comprising mRNA encoding dSpCas9-2xVP64 and gRNAs targeting VCAM1, ILR7a, and RUNX3. Results are shown for 72 hours post-transfection. FIG. 17A shows results for 72 hours post-transfection. FIG. 17B shows results for 120 hours pos- transfection.

[0086] FIG. 17C shows flow cytometry plots for assessing cell surface expression of VCAM1, Notch, or DLL4 in iHPCs transfected with LNPs for delivery of singleplex DNA- targeting systems comprising mRNA encoding dSpCas9-2xVP64 in combination with a guide targeting each gene. Results are shown for 120 hours post-transfection.

[0087] FIGS. 18A-18D shows a quantification of %DLL4+ cells and %VCAM1 cells as assessed by flow cytometry for iHPCs transfected with LNPs for delivery of mRNA encoding dSpCas9-2xVP64 and gRNAs targeting DLL4 and VCAM1 (DV) compared to a non-targeting gRNA (NT) control. Results are shown for D2, D7, D10 and D14 post-transfection. FIG. 18A shows a quantification of % DLL4+ cells for iHPCs transfected with a single dose of LNPs at DO. FIG. 18B shows a quantification of % VCAM1+ cells for iHPCs transfected with a single dose of LNPs at DO. FIG. 18C shows a quantification of %DLL4+ cells for iHPCs transfected with a dose of LNPs at DO and D8. FIG. 18D shows a quantification of % VCAM1+ cells for iHPCs transfected with a dose of LNPs at DO and D8.

[0088] FIG. 19A shows a timeline of iHPC differentiation into CD8+ mature T cells, with iHPCs at DO, CD4+ / CD8+ T-cell progenitor cells at D21 and CD8+ mature T cells at D28. FIG. 19B shows a quantification of % DLL4+ cells as assessed via flow cytometry for iHPCs transfected with various doses of LNPs for delivery of mRNA encoding dSpCas9-2xVP64 and a gRNA targeting DLL4 at DO. Results are shown for D2, D6, D9 and D14 post-transfection. Negative controls included cells transfected with LNPs encapsulating the dCas effector with a non-targeting guide RNA (NT).

[0089] FIGS. 19C-19F show results for DLL4 expression and cell viability for iHPCs transfected with various doses of LNPs for delivery of mRNA encoding dSpCas9-2xVP64 and a gRNA targeting DLL4 at DO. Results are shown for D2, D6, and D10 post-transfection. Negative controls included cells transfected with LNPs encapsulating the dCas effector without a guide (Cas9) or with a non-targeting guide RNA (NT). FIG. 19C shows a quantification of DLL4 surface protein expression as assessed by clow cytometry. FIG. 19D shows aquantification of DLL4 gene expression as assessed by RT-qPCR. Results are shown normalized to the GAPDH housekeeping gene and the Cas only control. FIG. 19E shows a quantification of live cells / mL as assessed by using a cell counter. FIG. 19F shows a quantification of %viability as assessed by flow cytometry.

[0090] FIG. 20A shows an experimental schematic and timeline of iHPC differentiation into CD56+ iNK cells. iHPCs generated from iPSCs were subjected to transcriptional activation using LNP-mediated delivery of a dSpCas9-2xVP64 mRNA and multiplexed guide RNAs at Day 0 and Day 7. Cells were assessed for cell surface marker expression at Day 21. Positive control cells were grown in plates coated with immobilized DLL4 and retronectin (DLL4 / RN). FIG. 20B shows representative flow plots assessing CD5+ / CD7+, CD4+CD8+, and CD56+ marker expression for the positive control and a DLL4+VCAM1 (DV) DNA-targeting system. FIG. 20C shows representative flow plots assessing CD5+ / CD7+, CD4+CD8+, and CD56+ marker expression for a DLL4+VCAM1+RUNX1 (DVR1) and a DLL4+VCAM1+RUNX3 (DVR3) DNA-targeting system.

[0091] FIG. 21A shows an experimental schematic and timeline of iHPC differentiation into DP CD4+ / CD8+ iT cell progenitors. iHPCs generated from iPSCs were subjected to transcriptional activation using LNP-mediated delivery of a dSpCas9-2xVP64 mRNA and multiplexed guide RNAs at Day 0 and Day 7. Cells were transferred to culture plates with a DLL4 / RN coating at D14 and cultured an additional seven days with the coating. Cells were assessed for cell surface marker expression at Day 21. FIG. 21B shows representative flow plots assessing CD5+ / CD7+, CD4+CD8+, and CD56+ marker expression for a DLL4+VCAM1 (DV) DNA-targeting system and a DLL4+VCAM1+RUNX3 (DVR3) DNA-targeting system.

[0092] FIG. 22A shows an experimental schematic and timeline of iHPC differentiation into induced common lymphoid progentiors (iCLPs). iHPCs generated from iPSCs were subjected to transcriptional activation using LNP-mediated delivery of a dSpCas9-2xVP64 mRNA and multiplexed guide RNAs at Day 4 and Day 7. Cells were assessed for cell surface marker expression at Day 14. FIG. 22B shows representative flow plots assessing CD45+, CD7+, and CD5+ / CD7+ marker expression for each multiplexed DNA-targeting system.

[0093] FIG. 23A shows an experimental schematic and timeline of iHPC differentiation into CD8+ iT cells using a hybrid Kaneko iT differentiation protocol comprising LNP delivery of a DNA-targeting system to iHPCs at Day 0 and Day 7 and culturing with a DLL4 / RN coating at D14 for 7 days. FIG. 23B shows an experimental schematic and timeline of iHPCdifferentiation into CD8+ iT cells using a standard Kaneko iT differentiation protocol comprising culturing iHPCs with a DLL4 / RN coating for 21 days without introduction of a DNA-targeting system. FIG. 23C shows a quantification of %CD45+ cells and %CD7+ / CD5+ cells, % CD4+ / CD8+ cells, %CD56+ cells gated from CD45+ cells as assessed by flow cytometry at D14 and D21 for cells introduced with a DLL4+VCAM1+RUNX3 (DVR3) DNA- targeting system and differentiated using the hybrid Kaneko protocol or cells cultured with the DLL4 / RN coating (Coating) using the standard Kaneko protocol without introduction of a DNA-targeting system. FIG. 23D shows an exemplary flow plot assessing CD8b and CD8a expression at Day 35 for cells delivered with a DVR3 DNA-targeting system and differentiated using the hybrid Kaneko protocol depicted in FIG. 23A. FIGS. 23E-23F show the results of a a serial tumor cell-killing co-culture assay. iT cells differentiated using the hybrid Kaneko protocol were cocultured with the HER2 antigen-expressing NSCLC cell line Hl 975 target cells at an effector cell ratio of 1:1 (FIG. 23E) or 1:50 (FIG. 23F). T cells without CAR specific to HER2 (Mock T) were used a negative control and HER2-CAR-T cells (CAR T) were used as a positive control.

[0094] FIG. 24A shows the results from a pooled screen for DLL4 targeting gRNAs. iHPCs were transduced with lentivirus comprising a pooled library of gRNAs followed by LNP delivery of dSpCas9-2xVP64 mRNA. DLL4+ cells were sorted at D2 and D6 post-LNP delivery and sequenced to identify the gRNAs that were enriched at D2 and / or D6. Results are shown for eleven gRNAs targeting the DLL4 TSS that were enriched at D2 and D6 with a log2 fold change in DLL4 expression greater than 4, in addition to the DLL4_5 gRNA used in the previous examples.

[0095] FIGS. 24B-24G show a quantification of DLL4 expression as assessed by flow cytometry for cells delivered with a DLL4 / VCAM1 (DV) DNA-targeting system each comprising a different DLL4 guide identified in a pooled screen. iHPCs were delivered with the DV DNA-targeting system at DO and were cultured in Kaneko differentiation media on plates without a DLL4 / RN. FIG. 24B shows a quantification of %DLL4+ cells at Day 2. FIG. 24C shows a quantification of %DLL4+ cells at Day 5. FIG. 24D shows a quantification of %DLL4+ cells at Day 7. FIG. 24E shows a quantification of %DLL4+ cells at Day 10. FIG. 24F shows DLL4 MFI as assessed by flow cytometry at Day 2, Day 5, Day 7, and Day 10. FIG. 24G shows DLL4 MFI as assessed by flow cytometry at Day 2, Day 5, Day 7, and Day 10 for a subset of the guides shown in FIG. 24F. 1

[0096] FIG. 25A shows an experimental schematic and timeline of iHPC differentiation into induced common lymphoid progenitors (iCLPs). iHPCs generated from iPSCs were subjected to transcriptional activation using LNP-mediated delivery of a dSpCas9-2xVP64 mRNA and multiplexed guide RNAs for targeting DLL4+VCAM1 (DV) and different combinations of LCD genes at Day 0 and Day 4. Each DV+LCD gene combination was tested using different DLL4 targeting gRNAs. FIGS. 25B-25G show a quantification of DLL4 expression as assessed by flow cytometry for cells delivered with multiplexed DV+LCD DNA-targeting systems comprising different DLL4 guides. FIG. 25B shows representative flow plots assessing DLL4 expression at Day 10 for cells delivered with DLL4+VCAM1+TCF7+BCL11B+MYB (DVTBM) DNA-targeting systems using the different DLL4 gRNAs. FIG. 25C shows a quantification of %DLL4+ cells as assessed by flow cytometry at Day 2, Day 7, Day 10, and Day 14 for each multiplexed DV+LCD DNA-targeting system tested comprising the different DLL4 guides. FIG. 25D shows a quantification of DLL4 MFI as assessed by flow cytometry at Day 2, Day 7, Day 10, and Day 14 for each multiplexed DV+LCD DNA-targeting system tested comprising the different DLL4 guides. FIG. 25E shows a quantification of %DLL4+ cells as assessed by flow cytometry at Day 2, Day 7, Day 10, and Day 14 for a subset of the multiplexed DV+LCD DNA-targeting systems and guides shown in FIG. 25C. FIG. 25F shows a quantification of DLL4 MFI as assessed by flow cytometry at Day 2, Day 7, Day 10, and Day 14 for a subset of the multiplexed DV+LCD DNA-targeting systems and guides shown in FIG.25D.

[0097] FIG. 25G shows a quantification of live cells / well as assessed using a cell counter at Day 4, Day 7, and Day 10 for cells delivered with multiplexed DV+LCD DNA-targeting systems comprising different DLL4 guides. FIG. 25H shows a quantification of % viability at Day 4, Day 7, and Day 10 for cells delivered with multiplexed DV+LCD DNA-targeting systems comprising different DLL4 guides.

[0098] FIG. 26A shows representative flow plots assessing CD5 and CD7 expression at D10 for cells delivered with DLL4+VCAM1+TCF7+BCL11B+MYB (DVTBM) DNA-targeting systems using three different DLL4 gRNAs compared to cells cultured with a DLL4 / RN coating (Coating). FIG. 26B shows representative flow plots assessing CD5 and CD7 expression at D10 for cells delivered with DLL4+VCAM1+TCF7+MYB+RUNX3 (DVTMR3) targeting systems using two different DLL4 gRNAs.

[0099] FIGS. 27A-27D shows a quantification of DLL4 expression as assessed by flow cytometry for iHPCs transfected with LNPs for delivery of (DLL4+VCAM1) DV DNA- targeting systems using two different guide RNAs. Results are shown for D2, D7, DIO and D14 post-transfection. FIG. 27A shows a quantification of % DLL4+ cells for iHPCs transfected with a single dose of LNPs at DO. FIG. 27B shows a quantification of DLL4 MFI for iHPCs transfected with a single dose of LNPs at DO. FIG. 27C shows a quantification of % DLL4+ cells for iHPCs transfected with a dose of LNPs at DO and D4. FIG. 27D shows a quantification of DLL4 MFI for iHPCs transfected with a dose of LNPs at DO and D4.

[0100] FIG. 28A shows an experimental schematic and timeline of iHPC differentiation into DP CD4+ / CD8+ iT cell progenitors using a hybrid Kaneko iT differentiation protocol. iHPCs generated from iPSCs were subjected to transcriptional activation using LNP-mediated delivery of a dSpCas9-2xVP64 mRNA and multiplexed guide RNAs at Day 0 and Day 4. Cells were transferred to culture plates with a DLL4 / RN coating at D14 and cultured an additional seven days with the coating. Cells were assessed for cell surface marker expression at Day 14 and Day 21. FIG. 28B shows representative flow plots assessing CD5+ / CD7+ marker expression at Day 14 for cells delivered with a DLL4+VCAM1+RUNX3 (DVR3) DNA-targeting system and differentiated using a hybrid Kaneko protocol. FIG. 28C shows representative flow plots assessing CD5+ / CD7+ and CD8+ / CD4+ marker expression at Day 21 for cells delivered with a DLL4+VCAM1+RUNX3 (DVR3) DNA-targeting system and differentiated using a hybrid Kaneko protocol.Detailed Description

[0101] Provided herein are DNA-targeting systems for promoting transcriptional activation of genes for promoting differentiation of hematopoietic progenitor cells (HPCs) into a differentiated population of cells, such as into lymphoid progenitor cells or lymphoid cells. In some embodiments, the DNA-targeting systems are based on epigenetic editing systems that promote the transcriptional activation of DLL4 and / or VCAM1. In some embodiments, one or more other lymphoid cell differentiation (LCD) also can be targeting for transcription activation using a provided DNA-targeting system. The DNA-binding systems include DNA-targeting modules that comprise a DNA-binding domain that binds to a target site for a gene as described (e.g., DLL4 or VCAM1), and (ii) at least one transcriptional activator effector domain. In some embodiments, the DNA-targeting systems include a plurality of DNA-targeting modules thateach target transcriptional activation to increase transcription of a gene. In some embodiments, the DNA-targeting systems include at least one DNA-targeting module for transcriptional activation of DLL4 and / or at least one DNA-targeting module for transcriptional activation of VCAM1. In some embodiments, the DNA-targeting systems a DNA-targeting module for transcriptional activation of DLL4 and a DNA-targeting module for transcriptional activation of VCAM1.

[0102] Provided herein is a DNA-targeting system in which the DNA-targeting system comprises a plurality of DNA-targeting modules, wherein the plurality of DNA-targeting modules comprises a first DNA-targeting module and a second DNA-targeting module. In some embodiments, the first DNA-targeting module increases transcription of a DLL4 gene. In some embodiments, the first DNA-targeting module comprises a fusion protein comprising (i) a DNA- binding domain that binds to a target site for DLL4, and (ii) at least one transcriptional activator effector domain. In some embodiments, the target site for DLL4 may be within a regulatory region, such as a promoter or enhancer of DLL4. In some embodiments, the second DNA- targeting module increases transcription of a VCAM1 gene. In some embodiments, the second DNA-targeting module comprises a fusion protein comprising (i) a DNA-binding domain that binds to a target site for VCAM1, and (ii) at least one transcriptional activator effector domain. In some embodiments, the target site for VCAM1 may be within a regulatory region, such as a promoter or enhancer of VCAM1.

[0103] In some embodiments, the DNA-targeting systems are synthetic transcription factors that can increase (or upregulate) transcription of a gene in a targeted manner. In some embodiments, the DNA-binding domain of the DNA-targeting system is a nuclease-inactive Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein (e.g., a dCas protein) or variant thereof complexed with a guide RNA (gRNA). Also provided are gRNAs for targeting to a target site in DLL4 or a regulatory DNA element thereof, or a target site in VC AMI or a regulatory DNA element thereof. Also provided are CRISPR-Cas / gRNA combinations thereof composed of the gRNA and a nuclease inactivated Cas, such as a dCas9. Also provided herein are polynucleotides encoding the DNA-targeting system or the fusion protein of the DNA-targeting system, and vectors and cells containing the same. Also provided herein are methods of using the DNA-targeting system for activating transcription of LCD genes in stem cells and promoting lymphoid cell differentiation.

[0104] In some embodiments, the DNA-targeting system includes a plurality of DNA-targeting modules, in which each DNA-targeting module is for targeting activation or increased expression of a different gene. In some embodiments, the DNA-targeting systems are multiplexed DNA-targeting systems, i.e. targeted to target sites for more than one gene. Hence, the terms DNA-targeting system may include a multiplexed DNA targeting system that includes more than one DNA-targeting module. A multiplexed DNA targeting system comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 DNA-targeting modules. In some embodiments, the plurality of DNA-targeting modules target activation of DLL4 and VCAM1. In some embodiments, the plurality of DNA targeting modules additionally target activation of 2 or more lymphoid cell differentiation (LCD) genes selected from MYB, RUNX3, RUNX1, SPI1, HEY1, IL7Ra, TBX21, LEF1, CBFB, TCF7, GATA3, and BCL1 IB. In some embodiments, the plurality of DNA targeting modules target activation of DLL4 and VCAM1 and one or more lymphoid cell differentiation (LCD) genes selected from MYB, RUNX3, RUNX1, SPI1, HEY1, IL7Ra, TBX21, LEF1, CBFB, TCF7, GATA3, and BCL11B.

[0105] In some embodiments, any two DNA-targeting modules of a DNA-targeting system comprise separate (i.e. non-overlapping) components. In some embodiments, different DNA- targeting modules of a DNA-targeting system comprise separate (i.e. non-overlapping) components. For example, a DNA-targeting system may comprise a first DNA-targeting module comprising a first fusion protein comprising a DNA-binding domain (e.g. a ZFN or TALE-based DNA-binding domain) that targets a first target site, and a second DNA-targeting module comprising a second fusion protein comprising a second DNA-binding domain (e.g. a ZFN or TALE-based DNA-binding domain) that targets a second target site.

[0106] In some embodiments, any two DNA-targeting modules of a DNA-targeting system may comprise shared (i.e. overlapping) components. In some embodiments, different DNA- targeting modules of a DNA-targeting system comprise shared (i.e. overlapping) components. For example, in one aspect, a DNA-targeting system may comprise a first DNA-targeting module comprising (a) a fusion protein comprising a Cas protein and a transcriptional effector (e.g. activation) domain, and (b) a first gRNA that complexes with the Cas protein and targets a first target site, and a second DNA-targeting module comprising (a) the fusion protein of the first DNA-targeting module, and (b) a second gRNA that complexes with the Cas protein and targets a second target site. It will be understood that providing two or more different gRNAs for a given Cas protein allows the same Cas protein to be targeted to the target sites of the two ormore gRNAs. Conversely, different Cas protein variants (e.g. SpCas9 and SaCas9) are compatible with different gRNA scaffold sequences and PAMs. Thus, it is possible to engineer a single DNA-targeting system comprising multiple non-overlapping CRISPR / Cas-based DNA- targeting modules.

[0107] The provided embodiments relate to compositions and methods for promoting differentiation of hematopoietic progenitor cells into a differentiated population of cells. In some embodiments, the differentiated population of cells are lymphoid progenitor cells. In some embodiments, the promoting occurs through transcriptional activation of DLL4 and VCAM1. In some embodiments, activating transcription of DLL4 and VCAM1 modulates the expression of cell-surface makers. In some embodiments, expression of a cell-surface marker is decreased, such as CD34 which is characteristic of hematopoietic progenitor cells. In some embodiments, expression of a cell-surface marker is decreased, such as CD 123 and / or CD 14, which are characteristic of monocytes. In some embodiments, expression of a cell-surface marker is increased, such as CD5, CD7, CD45RA, and / or CD 117, which are characteristic of lymphoid progenitor cells. In some embodiments, activation of DLL4 and VCAM1 genes promotes hematopoietic progenitor cell differentiation into lymphoid progenitor cells without the provision of extrinsic signaling ligands. In some embodiments, the methods can be used in connection with adoptive cell therapies.

[0108] In other aspects, the provided embodiments relate to compositions and methods for promoting differentiation of hematopoietic progenitor cells into a differentiated population of cells, wherein the differentiated population of cells are lymphoid cells (LCs). In some embodiments, the promoting occurs through transcriptional activation of DLL4 or VCAM1 along with one or more lymphoid cell differentiation (LCD) genes. In some embodiments, the promoting occurs through transcriptional activation of DLL4 and VCAM1 along with one or more lymphoid cell differentiation (LCD) genes. In some embodiments, the one or more lymphoid cell differentiation (LCD) genes are selected from MYB, RUNX3, RUNX1, SPI1, HEY1, IL7Ra, TBX21, LEF1, CBFB, TCF7, GATA3, and BCL11B. In some embodiments, LCs are differentiated from hematopoietic progenitor cells through any of the provided methods. In some embodiments, LCs are differentiated from lymphoid progenitor cells generated from hematopoietic progenitor cells through any of the provided methods. In some embodiments, a population comprising LCs is generated by culturing a population of hematopoietic progenitor cells under conditions to differentiate cells of the population of LCs. In some embodiments, apopulation comprising LCs is generated by culturing a population of lymphoid progenitor cells under conditions to differentiate cells of the population of LCs. In some embodiments, activating transcription of DLL4 and VCAM1 and / or one or more LCD genes modulates the expression of cell-surface makers. In some embodiments, expression of one or more cell-surface markers associated with Natural Killer (NK) cells is increased, such as CD56, CD45, CD16, DNAM, NKp30, and / or NKG2D. In some embodiments, expression of one or more cellsurface markers associated with T cells is increased, such as CD2, CD3, CD4, and / or CD8b. In some embodiments, activation of DLL4 and VCAM1 genes along with one or more LCD gene(s) promotes hematopoietic progenitor cell differentiation into lymphoid cells without the provision of extrinsic signaling ligands. In some embodiments, the methods can be used in connection with adoptive cell therapies.

[0109] The ability to differentiate various cell types from stem or progenitor cells such as hematopoietic progenitor cells has tremendous therapeutic potential, including in regenerative medicine and adoptive cell therapy. Current protocols for differentiating stem or progenitor cells into useful cell types and lineages, such as lymphoid cells, include providing extrinsic signals to stimulate differentiation. For example, hematopoietic progenitor cells are cultured in the presence of the Notch ligands (e.g. Delta-like protein 4 (DLL4) or Delta-like protein 1 (DLL1)), which can be provided immobilized on beads, coated on a surface, or expressed by feeder cells. The Delta ligands stimulate Notch signaling by inducing a mechanical shear force that catalyzes the release of the Notch receptor intracellular domain, which upregulates Notch target genes. Upregulation of Notch target genes (e.g., TCF7, GATA3, and BCL11B) promotes differentiation of stem cells, for example into lymphoid cells, such as NK cells or T cells.

[0110] However, provision of extrinsic Notch ligands such as DLL4 presents several challenges. Not only are the ligands costly and difficult to source, but the ligands must also be bound to a surface to induce the mechanical shear force necessary to stimulate Notch signaling. Since the ligands need to be coated on the surface of beads or microwells during cell culture, this makes scaling to larger cultivation systems (e.g., large-scale bioreactors) very difficult. Moreover, depleting the coated material from the culture poses an additional challenge.

[0111] The provided embodiments can be used to express Notch activating signals (i.e. DLL4 and VCAM1) within cells of the population of hematopoietic progenitor cells to be differentiated, thereby facilitating Notch signal activation without provisions for extrinsic Notch ligands. In particular, the provided embodiments provide for DNA-targeting systems andmethods that can be used to upregulate DLL4 and VCAM1 in hematopoietic progenitor cells and promote lymphoid progenitor cell differentiation. In some embodiments, the DNA-targeting systems and methods can be used to upregulate DLL4 and VC AMI in hematopoietic progenitor cells to promote lymphoid cell (LC) differentiation. Results herein demonstrate the surprising finding that the introduction of the provided DNA-targeting systems into stem or progenitor cells activates transcription of DLL4 and VCAM1 to biologically relevant levels. This approach circumvents the challenges associated with providing hematopoietic progenitor cells with extrinsic signaling ligands to stimulate differentiation. Moreover, the targeted gene activation does not modify DNA at the sequence level, thereby avoiding safety concerns with gene editing approaches. The ability to stimulate lymphoid progenitor cell differentiation using targeted gene activation provides an advantageous approach for producing highly functional cells for immunotherapy at scale.

[0112] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0113] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. DNA-TARGETING SYSTEMS

[0114] In some embodiments, provided are DNA-targeting systems capable of specifically targeting a target site for DLL4 and activating transcription of DLL4. In some embodiments, provided are DNA-targeting systems capable of specifically targeting a target site for VCAM1 and activating transcription of VCAM1. In some embodiments, provided are DNA-targeting systems capable of specifically targeting a target site for DLL4 and a target site for VCAM1, and activating transcription of DLL4 and VCAM1. In some embodiments, the target site for DLL4 is a target site in the gene or a regulatory DNA element thereof. In some embodiments, the target site for VC AMI is a target site in the gene or a regulatory DNA element thereof. In provided embodiments, the DNA-targeting system includes a fusion protein that comprises a DNA-binding domain that binds to the target site for the DLL4 gene and at least one effectordomain for activating transcription of the DLL4 gene. In further provided embodiments, the DNA-targeting system includes a fusion protein that comprises a DNA-binding domain that binds to the target site for the VC AMI gene and at least one effector domain for activating transcription of the VCAM1 gene. In some embodiments, the provided DNA-targeting systems increase transcription of DLL4 and / or VCAM1 in a hematopoietic progenitor cell. In some embodiments, transcriptional activation of gene expression by the DNA-targeting systems provided herein can promote lymphoid progenitor cell differentiation. In some embodiments, transcriptional activation of gene expression by the DNA-targeting systems provided herein can promote lymphoid cell (LC) differentiation.

[0115] In some embodiments, the at least one effector domain is a transcriptional activation domain for increasing transcription of one or more target genes. In some embodiments, the at least one effector domain increases transcription of DLL4 (e.g. activates or increases transcription of DLL4 as compared to transcription of the gene in the absence of the DNA- targeting system), such as any effector domain for transcriptional activation described in Section I.E. In some embodiments, the at least one effector domain increases transcription of VCAM1 (e.g. activates or increases transcription of VCAM1 as compared to transcription of the gene in the absence of the DNA-targeting system), such as any effector domain for transcriptional activation described in Section I.E.

[0116] In some embodiments, the effector domain directly or indirectly leads to increased transcription of the gene. In some embodiments, the effector domain induces, catalyzes or leads to transcription activation. In some embodiments, the effector domain induces transcription activation. In some aspects, the effector domain comprises: a VP64 domain, a p65 activation domain, a p300 domain, an Rta domain, a CBP domain, a VPR domain, a VPH domain, an HSF1 domain, a TET protein domain, optionally wherein the TET protein is TET1, a SunTag domain, or a domain, portion, variant, or truncation of any of the foregoing. In some embodiments, the effector domain is VP64. In some embodiments, the effector domain includes a NCOA3 domain, a FOXO3 domain or is a fusion of NCOA3 and FOXO3 domains. In some embodiments, such a fusion may further include a VP64 domain.

[0117] In some embodiments, the DNA-targeting system includes a fusion protein comprising (a) at least one DNA-binding domain capable of being targeted to the target site; and (b) at least one effector domain capable of modulating transcription of the gene. In some embodiments, the at least one effector domain is a transcriptional activation domain. The fusionprotein can be any suitable fusion protein, for example as described in Section I.F.

[0118] In some embodiments, the DNA-binding domain comprises or is derived from a CRISPR associated (Cas) protein, a zinc finger protein (ZFP), a transcription activator-like effector (TALE), meganuclease, homing endonuclease, LScel enzyme, or variants thereof. In some embodiments, the DNA-binding domain comprises a catalytically inactive (e.g. nucleaseinactive or nuclease-inactivated) variant of any of the foregoing. In some embodiments, the DNA-binding domain comprises a deactivated Cas9 (dCas9) protein or variant thereof that is a catalytically inactivated so that it is inactive for nuclease activity and is not able to cleave the DNA. The DNA-binding domain can be any suitable DNA-binding domain, for example as described in Sections I.C and I.D.

[0119] In some embodiments, the DNA-binding domain comprises or is derived from a Cas protein or variant thereof, such as a nuclease-inactive Cas or dCas (e.g. dCas9, and the DNA- targeting system comprises one or more guide RNAs (gRNAs), such as a combination of gRNAs (e.g. two gRNAs or three gRNAs). In some embodiments, the gRNA comprises a spacer sequence that is capable of targeting and / or hybridizing to the target site. In some embodiments, the gRNA is capable of complexing with the Cas protein or variant thereof. In some aspects, the gRNA directs or recruits the Cas protein or variant thereof to the target site. The gRNA can be any suitable gRNA, for example as described in section I.C.2.

[0120] In some embodiments, the DNA-targeting system is for increasing transcription of one or more genes, such as any described in Section I.B., and the fusion protein of a DNA- targeting module thereof is a dCas9-VP64 fusion protein, such as a dCas9-2xVP64 fusion protein. In some embodiments, the fusion protein is any as described herein, for example in Section I.F.

[0121] Exemplary components and features of the DNA-targeting systems are provided below in the following subsections.A. DNA-Targeting Modules and Multiplexed DNA-Targeting Systems

[0122] In some embodiments, the DNA-targeting system contains a DNA-targeting molecule capable of targeting a target site for a target gene. In some embodiments, the DNA- targeting system contains a plurality of DNA-targeting modules, where each DNA-targeting module of the plurality of DNA-targeting modules is a component of the DNA-targeting system that is independently capable of targeting a target site for a target gene. In provided embodiments, the target gene is DLL4 and / or VCAM1. In some embodiments, each DNA-targeting module includes (a) a DNA-binding domain capable of being targeted to the target site, and (b) an effector domain for modulating transcription of the gene. In some embodiments, the DNA-targeting system comprises a single DNA-targeting module for targeted transcriptional modulation of a single gene.

[0123] In some embodiments, the DNA-binding domain comprises: a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein; a zinc finger protein (ZFP); a transcription activator-like effector (TALE); a meganuclease; a homing endonuclease; or an I- Scel enzyme or a variant thereof, optionally wherein the DNA- binding domain comprises a catalytically inactive variant of any of the foregoing, wherein when the DNA-binding domain of each fusion protein comprises a Cas protein, the DNA-targeting system further comprises one or more gRNAs, each capable of targeting the Cas protein to a target site.

[0124] In some embodiments, a DNA-targeting module is a CRISPR / Cas-based DNA- targeting module. In some embodiments, in a CRISPR / Cas-based DNA-targeting module, the DNA-binding domain of the fusion protein is a Cas protein or variant thereof (e.g. a dCas protein, such as dCas9) and the DNA-targeting module further comprises a gRNA for targeting the DNA-binding domain to the target site.

[0125] In some embodiments, a DNA-targeting module is a zinc finger protein (ZFP) -based DNA-targeting module. In some embodiments, in a ZFP-based DNA-targeting module, the DNA-binding domain of the fusion protein is an engineered zinc finger protein (eZFP).

[0126] In some embodiments, a DNA-targeting module is a transcription activator-like effector (TALE) -based DNA-targeting module. In some embodiments, in a TALE-based DNA- targeting module, the DNA-binding domain of the fusion protein is an engineered TALE.

[0127] In some embodiments, the DNA-targeting system includes a plurality of DNA- targeting modules, in which each DNA-targeting module of the plurality of DNA-targeting modules targets a different target site. In some embodiments, one or more target sites are for different genes. In some embodiments, one or more target sites are for the same gene. In some embodiments, the DNA-targeting system is a multiplexed DNA-targeting system, i.e. is targeted to target sites for more than one gene. Hence, the term DNA-targeting system may include a multiplexed epigenetic-modifying DNA targeting system that includes more than one DNA- targeting module. In some embodiments, a multiplexed epigenetic-modifying DNA targeting system comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, or more DNA-targeting modules. In some embodiments, amultiplexed epigenetic-modifying DNA-targeting system comprises 2 DNA-targeting modules. In some embodiments, a multiplexed epigenetic-modifying DNA-targeting system comprises 3 DNA-targeting modules.

[0128] In some embodiments, any two DNA-targeting modules of a DNA-targeting system can comprise separate (i.e. non-overlapping) components. For example, a DNA-targeting system may comprise a first DNA-targeting module comprising a first fusion protein with a DNA- binding domain (e.g. a ZFN or TALE-based DNA-binding domain) that targets a first target site, and a second DNA-targeting module comprising a second fusion protein with a second DNA- binding domain (e.g. a ZFN or TALE-based DNA-binding domain) that targets a second target site.

[0129] In some embodiments, any two DNA-targeting modules of a DNA-targeting system can comprise shared (i.e. overlapping) components. For example, a DNA-targeting system may comprise: i) a first DNA-targeting module comprising (a) a fusion protein comprising a Cas protein and an effector domain, and (b) a first gRNA that complexes with the Cas protein and targets a first target site, and ii) a second DNA-targeting module comprising (a) the fusion protein of the first DNA-targeting module, and (b) a second gRNA that complexes with the Cas protein and targets a second target site. It will be understood that providing two or more different gRNAs for a given Cas protein allows the Cas protein to be targeted to the target sites of the two or more gRNAs. Conversely, different Cas protein variants (e.g. SpCas9 and SaCas9) are compatible with different gRNA scaffold sequences and PAMs, as described herein. Thus, it is possible to engineer a single DNA-targeting system comprising multiple non-overlapping CRISPR / Cas-based DNA-targeting modules.

[0130] In some aspects, provided herein is an epigenetic-modifying DNA-targeting system comprising a plurality of DNA-targeting modules for modulating transcription of one or more genes. In some embodiments, the plurality of DNA-targeting modules comprises a first DNA- targeting module for modulating transcription of a first gene of the one or more genes, and a second DNA-targeting module for modulating transcription of a second gene of the one or more genes. In some embodiments, the plurality of DNA-targeting modules comprises a first set of DNA-targeting modules for modulating transcription of a first set of genes, and a second set of DNA-targeting modules for modulating transcription of a second set of genes. In some embodiments, the first set of genes and the second set of genes comprise different genes. In some embodiments, the first set of genes and the second set of genes comprise overlappinggenes. In some embodiments, each DNA-targeting module comprises a fusion protein comprising: (a) a DNA-binding domain for targeting a target site of the target gene for the DNA-targeting module, and (b) at least one effector domain. In some embodiments, each DNA- targeting module comprises a transcriptional activator effector domain for increasing transcription of the one or more genes.B. Target Genes and Target Sites For Promoting Lymphoid Progenitor Cell Differentiation

[0131] In some aspects, the DNA-targeting systems target sites in DLL4 and VCAM1 genes, such as for transcriptional activation for increasing expression of DLL4 and / or VC AMI expression. In some embodiments, increasing expression of DLL4 and / or VCAM 1 in a cell, such as an HPC, promotes lymphoid progenitor or lymphoid cell differentiation. In some embodiments, the target site is targeted using any of the provided DNA-targeting systems.

[0132] In some embodiments, the DNA-targeting systems additionally target sites in one or more lymphoid cell differentiation (LCD) genes such as for transcriptional activation for increasing expression of the one or more LCD genes in combination with transcriptional activation of DLL4 and VCAM1. In embodiments as described, the one or more LCD genes are transcription factor genes. In some embodiments, increasing expression of DLL4 and / or VCAM 1 in combination with one or more other LCD genes in a cell, such as an HPC, promotes lymphoid progenitor or lymphoid cell differentiation. In some embodiments, the target sites are targeted using any of the provided DNA-targeting systems.

[0133] In some embodiments, the target site is targeted by a DNA-targeting system, such as by a DNA-targeting module of the DNA-targeting system, such as any described herein. In some embodiments, the target site for a gene is in the gene or a regulatory DNA element thereof. In some embodiments, provided herein are DNA-targeting systems that target either DLL4 or VCAM1 genes or regulatory DNA elements thereof described herein. In some embodiments, provided herein are multiplexed DNA-targeting systems that target both DLL4 and VCAM1 genes or regulatory DNA elements thereof described herein. In some embodiments, provided herein are multiplexed DNA-targeting systems that target both DLL4 and VCAM1 genes or regulatory elements thereof described herein, as well as one or more LCD genes or regulatory elements thereof described herein.

[0134] In some embodiments, the DNA-targeting system targets to or binds to a target site in a gene, such as any described herein. In some embodiments, the target site is located in the geneand / or a regulatory DNA element of the gene. In some embodiments, a regulatory DNA element is a sequence to which a gene regulatory protein may bind and affect transcription of the gene. In some embodiments, the regulatory DNA element is a cis, trans, distal, proximal, upstream, or downstream regulatory DNA element of a gene. In some embodiments, the regulatory DNA element is a promoter or enhancer of the gene. In some embodiments, the target site is located within a promoter, enhancer, exon, intron, untranslated region (UTR), 5’ UTR, or 3’ UTR of the gene. In some embodiments, the regulatory DNA element is a promoter. In some embodiments, a promoter is a nucleotide sequence to which RNA polymerase binds to begin transcription of the gene. In some embodiments, a promoter is a nucleotide sequence located within about lOObp, about 500bp, about lOOObp, or more, of a transcriptional start site (TSS) of the gene. In some embodiments, a promoter is within 500bp of a transcriptional start site of the gene. In some embodiments the target site is located within a sequence of unknown or known function that is suspected of being able to control expression of a gene.

[0135] In some embodiments, the DNA-targeting system targets to or binds within about 20bp within about 25bp, within about 50bp, within about 75bp, within about lOObp, within about 200bp within about 250bp, within about 300bp, within about 400bp, within about 500bp, within about, within about 600bp, 750bps, or within about lOOObp upstream of the transcriptional start site of the DLL4 gene. In some embodiments, the DNA-targeting system targets to or binds about 20bp- lOOObp upstream of the transcriptional start site of the DLL4 gene. In some embodiments, the DNA-targeting system targets to or binds within about 20bp upstream of the transcriptional start site of the DLL4 gene. In some embodiments, the DNA- targeting system targets to or binds within about 25bp upstream of the transcriptional start site of the DLL4 gene. In some embodiments, the DNA-targeting system targets to or binds within about 50bp upstream of the transcriptional start site of the DLL4 gene. In some embodiments, the DNA-targeting system targets to or binds within about 75bp upstream of the transcriptional start site of the DLL4 gene. In some embodiments, the DNA-targeting system targets to or binds within about lOObp upstream of the transcriptional start site of the DLL4 gene. In some embodiments, the DNA-targeting system targets to or binds within about 200bp upstream of the transcriptional start site of the DLL4 gene. In some embodiments, the DNA-targeting system targets to or binds within about 250bp upstream of the transcriptional start site of the DLL4 gene. In some embodiments, the DNA-targeting system targets to or binds within about 300bp upstream of the transcriptional start site of the DLL4 gene. In some embodiments, the DNA-targeting system targets to or binds within about 400bp upstream of the transcriptional start site of the DLL4 gene. In some embodiments, the DNA-targeting system targets to or binds within about 500bp upstream of the transcriptional start site of the DLL4 gene. In some embodiments, the DNA-targeting system targets to or binds within about 600bp upstream of the transcriptional start site of the DLL4 gene. In some embodiments, the DNA-targeting system targets to or binds within about 750bp upstream of the transcriptional start site of the DLL4 gene. In some embodiments, the DNA-targeting system targets to or binds within about lOOObp upstream of the transcriptional start site of the DLL4 gene. In some embodiments, the target site is within the promoter of the DLL4 gene.

[0136] In some embodiments, the DNA-targeting system targets to or binds within about 20bp, within about 25bp, within about 50bp, within about 75bp, within about lOObp, within about 200bp, within about 250bp, within about 300bp, within about 400bp, within about 500bp, within about 600bp, within about, 750bps, or within about lOOObp upstream of the transcriptional start site of the VCAM1 gene. In some embodiments, the DNA-targeting system targets to or binds about 20bp- lOOObp upstream of the transcriptional start site of the VCAM1 gene. In some embodiments, the DNA-targeting system targets to or binds within about 20bp upstream of the transcriptional start site of the VCAM1 gene. In some embodiments, the DNA- targeting system targets to or binds within about 25bp upstream of the transcriptional start site of the VCAM1 gene. In some embodiments, the DNA-targeting system targets to or binds within about 50bp upstream of the transcriptional start site of the VCAM1 gene. In some embodiments, the DNA-targeting system targets to or binds within about 75bp upstream of the transcriptional start site of the VCAM1 gene. In some embodiments, the DNA-targeting system targets to or binds within about lOObp upstream of the transcriptional start site of the VCAM1 gene. In some embodiments, the DNA-targeting system targets to or binds within about 200bp upstream of the transcriptional start site of the VCAM1 gene. In some embodiments, the DNA-targeting system targets to or binds within about 250bp upstream of the transcriptional start site of the VCAM1 gene. In some embodiments, the DNA-targeting system targets to or binds within about 300bp upstream of the transcriptional start site of the VCAM1 gene. In some embodiments, the DNA- targeting system targets to or binds within about 400bp upstream of the transcriptional start site of the VCAM1 gene. In some embodiments, the DNA-targeting system targets to or binds within about 500bp upstream of the transcriptional start site of the VCAM1 gene. In some embodiments, the DNA-targeting system targets to or binds within about 600bp upstream of thetranscriptional start site of the VCAM1 gene. In some embodiments, the DNA-targeting system targets to or binds within about 750bp upstream of the transcriptional start site of the VCAM1 gene. In some embodiments, the DNA-targeting system targets to or binds within about lOOObp upstream of the transcriptional start site of the VCAM1 gene. In some embodiments, the target site is within the promoter of the VCAM1 gene.

[0137] In some embodiments, the DNA-targeting system targets to or binds within about 20bp, within about 25bp, within about 50bp, within about 75bp, within about lOObp, within about 200bp, within about 250bp, within about 300bp, within about 400bp, within about 500bp, within about 600bp, within about, 750bps, or within about lOOObp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds about 20bp- lOOObp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds within about 20bp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds within about 25bp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds within about 50bp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA- targeting system targets to or binds within about 75bp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds within about lOObp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds within about 200bp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds within about 250bp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds within about 300bp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds within about 400bp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds within about 500bp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA- targeting system targets to or binds within about 600bp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds within about 750bp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds within about lOOObp upstream of the transcriptional start site of the LCD gene. In some embodiments, the target site is within thepromoter of the LCD gene.J. DLL4 and VCAMJ genes and target sites

[0138] In some embodiments, delivery of the DNA-targeting system increases transcription of the DLL4 gene. In some embodiments, delivery of the DNA-targeting system increases transcription of the VCAM1 gene. In some embodiments, provided herein are target sites for DLL4 for which increased transcription promotes lymphoid progenitor differentiation and / or lymphoid cell differentiation. In some embodiments, provided herein are target sites for VCAM1 for which increased transcription promotes lymphoid progenitor differentiation and / or lymphoid cell differentiation.

[0139] In some embodiments, the DNA-targeting system comprises a plurality of DNA- targeting modules. In some embodiments, each DNA-targeting module of the plurality of DNA- targeting modules targets a target site. In some embodiments, the plurality of DNA-targeting modules target at least DLL4. In some embodiments, the plurality of DNA-targeting modules target at least VCAM1. In some embodiments, the plurality of DNA-targeting modules target DLL4 and VCAM1.

[0140] In some embodiments, the target site for DLL4 is located with within the genomic coordinates of human genome assembly GRCh38 (hg38) chrl5: 40,905,000 to chrl 5:40,909,000. In some embodiments, the target site for DLL4 is located with within the genomic coordinates of human genome assembly GRCh38 (hg38) chrl5: 40,905,204 to chrl5:40,908, 702. In some embodiments, the target site for DLL4 is located with within the genomic coordinates of human genome assembly GRCh38 (hg38) chrl5: 40,937,800 to chrl5:40,938,800. In some embodiments, the target site for DLL4 is located with within the genomic coordinates of human genome assembly GRCh38 (hg38) chrl5: 40,937,844 to chrl5:40,938,701. In some embodiments, the target site for DLL4 is located with within the genomic coordinates of human genome assembly GRCh38 (hg38) chrl5: 40,940,000 to chrl5:40,943,500. In some embodiments, the target site for DLL4 is located with within the genomic coordinates of human genome assembly GRCh38 (hg38) chrl5: 40,940,247 to chrl5:40,943,253.

[0141] In some embodiments, the target site for DLL4 is located with within the genomic coordinates of human genome assembly GRCh38 (hg38) chrl5: 40,925,250 to chrl5:40,934,00. In some embodiments, the target site for DLL4 is located with within the genomic coordinates of human genome assembly GRCh38 (hg38) chrl5: 40,925,621 to chrl5:40,933, 969.

[0142] In some embodiments, the target site for DLL4 is located within 1000 bp of human genome assembly GRCh38 (hg38) genomic coordinates chrl5: 40,929,340. In some embodiments, the target site for DLL4 is located with within the genomic coordinates of human genome assembly GRCh38 (hg38) chrl5: 40,928,340 to chrl5:40,930,340. In some embodiments, the target site for DLL4 is located within 550bp of human genome assembly GRCh38 (hg38) genomic coordinates chrl5: 40,929,340. In some embodiments the target site for DLL4 is located within the genomic coordinates human genome assembly GRCh38 (hg38) chrl5: 40,928,840 to 40,929,840. In some embodiments, the target site for DLL4 is located within 250bp of human genome assembly GRCh38 (hg38) genomic coordinates chrl5: 40,929,340. In some embodiments the target site for DLL4 is located within the genomic coordinates human genome assembly GRCh38 (hg38) chrl5: 40,929,090 to 40,929,590. In some embodiments the target site for DLL4 is located within the genomic coordinates human genome assembly GRCh38 (hg38) chrl5: 40,929,240- 40,929,440. In some embodiments the target site for DLL4 is located within the genomic coordinates human genome assembly GRCh38 (hg38) chrl5: 40,929,290 to 40,929,390. In some embodiments the target site for DLL4 is located within the genomic coordinates human genome assembly GRCh38 (hg38) chrl5: 40,929,100 to chrl5:40,929,170. In some embodiments the target site for DLL4 is located within the genomic coordinates human genome assembly GRCh38 (hg38) chrl5: 40,929,130 to chrl5:40,929,149. In some embodiments the target site for DLL4 is located within the genomic coordinates human genome assembly GRCh38 (hg38) chrl5: 40,929,120 to chrl5:40,929,139.

[0143] In some embodiments, the target site for DLL4 comprises a sequence selected from any one of SEQ ID NOs: 1-2 and 147-166, or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing. In some embodiments, the target site for DLL4 is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a target site sequence of any one of SEQ ID NOS: 1-2 and 147-166. In some embodiments, the target site for DLL4 is a contiguous portion of any one of SEQ ID NOS: 1-2 and 147-166.

[0144] In some embodiments, the target site for DLL4 comprises a sequence selected from any one of SEQ ID NO: 1-2 or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing. In some embodiments, the target site for DLL4 is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a target site sequence of any one of SEQ ID NOS: 1-2. In some embodiments, the target site for DLL4 is a contiguous portion of any one of SEQ ID NOS: 1-2.

[0145] In some embodiments, the target site for DLL4 comprises a sequence selected from any one of SEQ ID NO: 1-2 or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing. In some embodiments, the target site for DLL4 is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a target site sequence of any one of SEQ ID NOS: 1-2. In some embodiments, the target site for DLL4 is a contiguous portion of any one of SEQ ID NOS: 1-2.

[0146] In some embodiments, the target site for DLL4 comprises the sequence set forth in SEQ ID NO: 154, or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing. In some embodiments, the target site for DLL4 is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a target site sequence set forth in SEQ ID NO: 154. In some embodiments, the target site for DLL4 is a contiguous portion SEQ ID NO: 154.

[0147] In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO:1. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO:2. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 147. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 148. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 149. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 150. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 151. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 152. In some embodiments, the DNA- targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 153. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 154. In some embodiments, the DNA-targeting systemtargets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 155. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 156. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 157. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 158. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 159. In some embodiments, the DNA- targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 160. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 161. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 162. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 163. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 164. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 165. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 166.

[0148] In some embodiments, the target site for VCAM1 is located within 1000 bp of human genome assembly GRCh38 (hg38) genomic coordinates chrl: 100,719,742. In some embodiments, the target site for VCAM1 is located with within the genomic coordinates of human genome assembly GRCh38 (hg38) chrl: 100,718,742 to chrl: 100,720,742. In some embodiments, the target site for VCAM1 is located within 550bp of human genome assembly GRCh38 (hg38) genomic coordinates chrl: 100,719,742. In some embodiments the target site for VCAM1 is located within the genomic coordinates human genome assembly GRCh38 (hg38) chrl: 100,719,182 to chrl: 100,720,290. In some embodiments the target site for VCAM1 is located within the genomic coordinates human genome assembly GRCh38 (hg38) chrl: 100,719,242 to 100,720,242. In some embodiments, the target site for VCAM1 is located within 250bp of human genome assembly GRCh38 (hg38) genomic coordinates chrl: 100,719,742. In some embodiments the target site for VCAM1 is located within the genomic coordinates human genome assembly GRCh38 (hg38) chrl: 100,719,492 to 100,719,992. In some embodiments the target site for VCAM1 is located within the genomic coordinates human genome assembly GRCh38 (hg38) chrl: 100,719,642- 100,719,842. In some embodiments the target site forVCAM1 is located within the genomic coordinates human genome assembly GRCh38 (hg38) chrl: 100,719,692 to 100,719,792. In some embodiments the target site for DLL4 is located within the genomic coordinates human genome assembly GRCh38 (hg38) chrl5: 40,929,100 to chrl5:40,929,170. In some embodiments the target site for VCAM1 is located within the genomic coordinates human genome assembly GRCh38 (hg38) chrl: 100,719,492 to chrl: 100,719,742.

[0149] In some embodiments, the target site for VCAM1 comprises a sequence selected from any one of SEQ ID NO: 3-7 or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing. In some embodiments, the target site for VCAM1 is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a target site sequence of any one of SEQ ID NOS: 3-7. In some embodiments, the target site for VCAM1 is a contiguous portion of any one of SEQ ID NOS: 3-7.

[0150] In some embodiments, the target site for VCAM1 comprises the sequence set forth in SEQ ID NO: 3 or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing. In some embodiments, the target site for VCAM1 is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a target site sequence of SEQ ID NO: 3. In some embodiments, the target site for VCAM1 is a contiguous portion of SEQ ID NO: 3.

[0151] In some embodiments, the DNA-targeting system targets a target site for VCAM1 comprising the sequence set forth in SEQ ID NOG. In some embodiments, the DNA-targeting system targets a target site for VCAM1 comprising the sequence set forth in SEQ ID NO:4. In some embodiments, the DNA-targeting system targets a target site for VCAM1 comprising the sequence set forth in SEQ ID NOG. In some embodiments, the DNA-targeting system targets a target site for VCAM1 comprising the sequence set forth in SEQ ID NOG. In some embodiments, the DNA-targeting system targets a target site for VCAM1 comprising the sequence set forth in SEQ ID NOG.2. DLL4 / VCAM1 and LCD genes and target sites

[0152] In some embodiments, delivery of the DNA-targeting system increases transcription of the DLL4 gene, the VC AMI gene and one or more LCD genes selected from the groupconsisting of: MYB, RUNX3, RUNX1, SPI1, HEY1, IL7Ra, TBX21, LEF1, CBFB, TCF7, GATA3, and BCL1 IB. In some embodiments, provided herein are target sites for DLL4, target sites for VCAM1, and target sites for one or more LCD genes, for which increased transcription promotes lymphoid progenitor differentiation and / or lymphoid cell differentiation.

[0153] . In some embodiments, delivery of the DNA-targeting system increases transcription of the DLL4 gene, the VC AMI gene, and one or more LCD genes selected from the group consisting of: RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL11B. In some embodiments, delivery of the DNA-targeting system increases transcription of the DLL4 gene, the VCAM1 gene, and one or more LCD genes selected from the group consisting of: RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1. In some embodiments, delivery of the DNA-targeting system increases transcription of the DLL4 gene, the VCAM1 gene, and any combination of three different LCD genes selected from the group consisting of: RUNX3, IL7Ra, TBX21, LEF1, MYB, RUNX1, SPI1, and HEY1. In some embodiments, delivery of the DNA-targeting system increases transcription of the DLL4 gene, the VC AMI gene, and RUNX3 and IL7Ra, and one or more LCD genes selected from the group consisting of: RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some embodiments, delivery of the DNA-targeting system increases transcription of the DLL4 gene, the VC AMI gene, and RUNX3, IL7Ra, and TBX21, and one or more LCD genes selected from the group consisting of: RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some embodiments, delivery of the DNA-targeting system increases transcription of the DLL4 gene, the VCAM1 gene, and RUNX3, IL7Ra, and CBFB, and one or more LCD genes selected from the group consisting of: RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some embodiments, delivery of the DNA-targeting system increases transcription of the DLL4 gene, the VCAM1 gene, and LCD genes in any of the combinations listed in Table E6. In some embodiments, delivery of the DNA-targeting system increases transcription of the DLL4 gene, the VC AMI gene, and one or more LCD genes selected from the group consisting of: TCF7, GATA3, and BCL11B.

[0154] In some embodiments, the DNA-targeting system comprises a plurality of DNA- targeting modules. In some embodiments, each DNA-targeting module of the plurality of DNA- targeting modules targets a target site. In some embodiments, the plurality of DNA-targeting modules target at least DLL4, VCAM1, and one or more LCD genes selected from the groupconsisting of MYB, RUNX3, RUNX1, SPI1, HEY1, IL7Ra, TBX21, LEF1, CBFB, TCF7, GATA3, and BCL11B.

[0155] In some embodiments, the plurality of DNA-targeting modules target DLL4, VCAM1, and one or more LCD genes selected from the group consisting of RUNX1, RUNX3, and LEF1. In some embodiments, the plurality of DNA-targeting modules target DLL4, VCAM1, and RUNX1. In some embodiments, the plurality of DNA-targeting modules target DLL4, VCAM1, and RUNX3. In some embodiments, the plurality of DNA-targeting modules target DLL4, VCAM1, and LEF1.

[0156] In some embodiments, the plurality of DNA-targeting modules target DLL4, VCAM1, and one or more LCD genes selected from the group consisting of TCF7, MYB, BCL11B, RUNX3, GATA3, and IL7Ra. In some embodiments, the plurality of DNA-targeting modules target DLL4, VCAM1, TCF7, MYB, and BCL11B. In some embodiments, the plurality of DNA-targeting modules target DLL4, VCAM1, TCF7, MYB, and RUNX3. In some embodiments, the plurality of DNA-targeting modules target DLL4, VCAM1, and BCL11B. In some embodiments, the plurality of DNA-targeting modules target DLL4, VCAM1, TCF7, and BCL11B. In some embodiments, the plurality of DNA-targeting modules target DLL4, VCAM1, TCF7, BCL11B, and GATA3.

[0157] In some embodiments, the DNA-targeting system targets a target site for DLL4, VCAM1 and one or more LCD genes. In some embodiments, the DNA-targeting system targets a target site for DLL4, a target site for VCAM1, and a target site for one or more LCD genes selected from the group consisting of MYB, RUNX3, RUNX1, SPI1, HEY1, IL7Ra, TBX21, LEF1, CBFB, TCF7, GATA3, and BCL11B.

[0158] In some embodiments, the DNA-targeting system targets a target site for DLL4, VCAM1, and one or more LCD genes selected from the group consisting of RUNX1, RUNX3, and LEF1. In some embodiments, the DNA-targeting system targets a target site for DLL4, VCAM1, and RUNX1. In some embodiments the DNA-targeting system targets a target site for DLL4, VCAM1, and RUNX3. In some embodiments, the DNA-targeting system targets a target site for DLL4, VCAM1, and LEF1.

[0159] In some embodiments, the DNA-targeting system targets a target site for DLL4, VCAM1, and one or more LCD genes selected from the group consisting of TCF7, MYB, BCL11B, RUNX3, GATA3, and IL7Ra. In some embodiments, the DNA-targeting system targets a target site for DLL4, VCAM1, TCF7, MYB, and BCL11B. In some embodiments, theDNA-targeting system targets a target site for DLL4, VCAM1, TCF7, MYB, and RUNX3. In some embodiments, the DNA-targeting system targets a target site for DLL4, VCAM1, and BCL1 IB. In some embodiments, the DNA-targeting system targets a target site for DLL4, VCAM1, TCF7, and BCL11B. In some embodiments the DNA-targeting system targets a target site for DLL4, VCAM1, TCF7, BCL11B, and GATA3.

[0160] In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VC AMI gene, and one or more LCD genes, wherein the one or more LCD genes are selected from the group consisting of MYB, RUNX3, RUNX1, SPI1, HEY1, IL7Ra, TBX21, LEF1, CBFB, TCF7, GATA3, and BCL11B. In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VCAM1 gene, and 2 or more of the following LCD genes: MYB, RUNX3, RUNX1, SPI1, HEY1, IL7Ra, TBX21, LEF1, CBFB, TCF7, GATA3, and BCL1 IB. In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VC AMI gene, and 3 or more of the following LCD genes: MYB, RUNX3, RUNX1, SPI1, HEY1, IL7Ra, TBX21, LEF1, CBFB, TCF7, GATA3, and BCL11B. In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VCAM1 gene, and 4 or more of the following LCD genes: MYB, RUNX3, RUNX1, SPI1, HEY1, IL7Ra, TBX21, LEF1, CBFB, TCF7, GATA3, and BCL11B. In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VCAM1 gene, and 5 or more of the following LCD genes: MYB, RUNX3, RUNX1, SPI1, HEY1, IL7Ra, TBX21, LEF1, CBFB, TCF7, GATA3, and BCL1 IB. In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VCAM1 gene, and 6 or more of the following LCD genes: MYB, RUNX3, RUNX1, SPI1, HEY1, IL7Ra, TBX21, LEF1, CBFB, TCF7, GATA3, and BCL11B.

[0161] In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VC AMI gene, and one or more LCD genes, wherein the one or more LCD genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1. In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VC AMI gene, and 2 or more of the following LCD genes: RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1. In some embodiments, the DNA- targeting system targets a target site for the DLL4 gene, the VCAM1 gene, and 3 or more of the following LCD genes: RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1. In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VC AMI gene, and 4 or more of the following LCD genes: RUNX3, IL7Ra, TBX21, CBFB,LEF1, MYB, RUNX1, SPI1, and HEY1. In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VCAM1 gene, and 5 or more of the following LCD genes: RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1. In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VCAM1 gene, and 6 or more of the following LCD genes: RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1.

[0162] In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VC AMI gene, and any combination of three different LCD genes selected from the group consisting of: RUNX3, IL7Ra, TBX21, LEF1, MYB, RUNX1, SPI1, and HEY1. In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VCAM1 gene, and a first LCD gene, a second LCD gene and a third LCD gene. In some embodiments the first, second, and third LCD gene are independently selected from the group consisting of RUNX3, IL7Ra, TBX21, LEF1, MYB, RUNX1, SPI1, and HEY1. In some embodiments, the first and second LCD gene are RUNX3 and IL7Ra and the third LCD gene is selected from the group consisting of: TBX21, LEF1, MYB, RUNX1, SPI1, and HEY1.

[0163] In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VC AMI gene, and 2 or more of the following LCD genes: RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VCAM1 gene, and 3 or more of the following LCD genes: RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VCAM1 gene, and 4 or more of the following LCD genes: RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some embodiments, the DNA- targeting system targets a target site for the DLL4 gene, the VCAM1 gene, and 5 or more of the following LCD genes: RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VCAM1 gene, and 6 or more of the following LCD genes: RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VCAM1 gene, and each of the following LCD genes: RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

[0164] In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VC AMI gene, and RUNX3, IL7Ra, and TBX21, and one or more LCD genes selected from the group consisting of: RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In someembodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VCAM1 gene, and RUNX3, IL7Ra, and CBFB, and one or more LCD genes selected from the group consisting of: RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VCAM1 gene, and RUNX3 and IL7Ra, and one or more LCD genes selected from the group consisting of: RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VCAM1 gene, and any of the combinations of LCD genes listed in Table E6.

[0165] In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VC AMI gene, and each of the following LCD genes: RUNX3 and IL7Ra. In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VCAM1 gene, and each of the following LCD genes: RUNX3, IL7Ra, and CBFB. In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VCAM1 gene, and each of the following LCD genes: RUNX3, IL7Ra, and TBX21. In some embodiments, the DNA- targeting system targets a target site for the DLL4 gene, the VCAM1 gene, and each of the following LCD genes: RUNX3, CBFB, LEF1, MYB, TBX21, and IL7Ra. In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VCAM1 gene, and each of the following LCD genes: RUNX3, CBFB, LEF1, MYB, and TBX21. In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VCAM1 gene, and each of the following LCD genes: RUNX3, CBFB, LEF1, and MYB. In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VCAM1 gene, and each of the following LCD genes: LEF1, MYB, and TBX21. In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VCAM1 gene, and each of the following LCD genes: LEF1 and TBX21. In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VCAM1 gene, and each of the following LCD genes: LEF1 and MYB.

[0166] In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VC AMI gene, and one or more LCD genes, wherein the one or more LCD genes are selected from the group consisting of TCF7, GATA3, and BCL1 IB. In some embodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VCAM1 gene, and at least a first LCD gene and a second LCD gene, wherein the first and second LCD gene are independently selected from the group consisting of TCF7, GATA3, and BCL11B. In someembodiments, the DNA-targeting system targets a target site for the DLL4 gene, the VCAM1 gene, and TCF7, GATA3, and BCL11B.

[0167] In some embodiments, the target site for DLL4 comprises a sequence selected from any one of SEQ ID NOs: 1-2 and 147-166, or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing, the target site for VCAM1 comprises a sequence selected from any one of SEQ ID NOs: 3-7 or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing, and the one or more LCD genes comprises a sequence selected from any one of SEQ ID NOS: 40-53 and SEQ ID NOS: 93-110, or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing.

[0168] In some embodiments, the target site for DLL4 comprises a sequence selected from any one of SEQ ID NOs: 1-2 or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing, the target site for VCAM1 comprises a sequence selected from any one of SEQ ID NOs: 3-7 or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing, and the target site for one or more LCD genes comprises a sequence selected from any one of SEQ ID NOS: 40-53 and SEQ ID NOS: 93-110, or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing.

[0169] In some embodiments, the target site for DLL4 comprises a sequence selected from SEQ ID NOs: 2 or SEQ ID NO: 154 or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing, the target site for VC AMI comprises the sequence set forth in SEQ ID NO: 3 or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of thereof, and the target site for one or more LCD genes comprises a sequence selected from any one of SEQ ID NOS: 40-53 and SEQ ID NOS: 93-110, or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing.

[0170] In some embodiments, the target site for DLL4 comprises the sequence set forth in SEQ ID NO: 154 or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the thereof, the target site for VC AMI comprises the sequence set forth in SEQ ID NO: 3 or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing, and the target site for one or more LCD genes comprises thesequence set forth in SEQ ID NO: 46 or SEQ ID NO: 47 , or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing.

[0171] In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOS: 1-2 and 147-176, SEQ ID NOS: 3-7, SEQ ID NOS: 40-53 and SEQ ID NOS: 93-110 that is 15, 16, 17, 18 or 19 nucleotides in length, or a complementary sequence of any of the foregoing. In some embodiments, the target site is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a target site sequence described herein above. In some embodiments, the target site for DLL4 is the sequence set forth in any one of SEQ ID NOS: 1-2 and 147-176, the target site for VCAM1 is the sequence set forth in any one of SEQ ID NOS: 3- 7, and the target site for the one or more LCD genes is the sequence set forth in any one of SEQ ID NOS: 40-53 and SEQ ID NOS: 93-110.

[0172] In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO:1. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO:2. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 147. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 148. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 149. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 150. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 151. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 152. In some embodiments, the DNA- targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 153. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 154. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 155. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 156. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 157. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQID NO: 158. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 159. In some embodiments, the DNA- targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 160. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 161. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 162. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 163. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 164. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 165. In some embodiments, the DNA-targeting system targets a target site for DLL4 comprising the sequence set forth in SEQ ID NO: 166.

[0173] In some embodiments, the DNA-targeting system targets a target site for VCAM1 comprising the sequence set forth in SEQ ID NO:3. In some embodiments, the DNA-targeting system targets a target site for VCAM1 comprising the sequence set forth in SEQ ID NO:4. In some embodiments, the DNA-targeting system targets a target site for VCAM1 comprising the sequence set forth in SEQ ID NO:5. In some embodiments, the DNA-targeting system targets a target site for VCAM1 comprising the sequence set forth in SEQ ID NO:6. In some embodiments, the DNA-targeting system targets a target site for VCAM1 comprising the sequence set forth in SEQ ID NO:7.

[0174] In some embodiments, the DNA-targeting system targets a target site for MYB. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 40 or SEQ ID NO: 41, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 40 or SEQ ID NO: 41, or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 40 or SEQ ID NO: 41. In some embodiments, the DNA-targeting system targets a target site for MYB comprising the sequence set forth in SEQ ID NO:40. In some embodiments, the DNA- targeting system targets a target site for MYB comprising the sequence set forth in SEQ ID NO:41.

[0175] In some embodiments, the DNA-targeting system targets a target site for RUNX1. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 42 or SEQID NO: 43, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 42 or SEQ ID NO: 43, or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 42 or SEQ ID NO: 43. In some embodiments, the DNA-targeting system targets a target site for RUNX1 comprising the sequence set forth in SEQ ID NO:42. In some embodiments, the DNA- targeting system targets a target site for RUNX1 comprising the sequence set forth in SEQ ID NO:43.

[0176] In some embodiments, the DNA-targeting system targets a target site for HEY1. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 44 or SEQ ID NO: 45, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 44 or SEQ ID NO: 44, or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 44 or SEQ ID NO: 45. In some embodiments, the DNA-targeting system targets a target site for HEY1 comprising the sequence set forth in SEQ ID NO:44. In some embodiments, the DNA- targeting system targets a target site for HEY 1 comprising the sequence set forth in SEQ ID NO:45.

[0177] In some embodiments, the DNA-targeting system targets a target site for RUNX3. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 46 or SEQ ID NO: 47, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 46 or SEQ ID NO: 47, or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 46 or SEQ ID NO: 47. In some embodiments, the DNA-targeting system targets a target site for RUNX3 comprising the sequence set forth in SEQ ID NO:46. In some embodiments, the DNA- targeting system targets a target site for RUNX3 comprising the sequence set forth in SEQ ID NO:47.

[0178] In some embodiments, the DNA-targeting system targets a target site for SPI1. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 48 or SEQ ID NO: 49, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequenceset forth in SEQ ID NO: 48 or SEQ ID NO: 49, or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 48 or SEQ ID NO: 49. In some embodiments, the DNA-targeting system targets a target site for SPI1 comprising the sequence set forth in SEQ ID NO:48. In some embodiments, the DNA- targeting system targets a target site for SPI1 comprising the sequence set forth in SEQ ID NO:49.

[0179] In some embodiments, the DNA-targeting system targets a target site for IL7Ra. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 50, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 50, or a complementary sequence of any of the foregoing. In some embodiments, the DNA- targeting system targets a target site for IL7Ra comprising the sequence set forth in SEQ ID NO:50.

[0180] In some embodiments, the DNA-targeting system targets a target site for TBX21. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 51, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 51, or a complementary sequence of any of the foregoing. In some embodiments, the DNA- targeting system targets a target site for TBX21 comprising the sequence set forth in SEQ ID NO: 51.

[0181] In some embodiments, the DNA-targeting system targets a target site for LEF1. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 52, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 52, or a complementary sequence of any of the foregoing. In some embodiments, the DNA- targeting system targets a target site for LEF1 comprising the sequence set forth in SEQ ID NO:52.

[0182] In some embodiments, the DNA-targeting system targets a target site for CBFB. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 53, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 53, or a complementary sequence of any of the foregoing. In some embodiments, the DNA-targeting system targets a target site for CBFB comprising the sequence set forth in SEQ ID NO:53.

[0183] In some embodiments, the DNA-targeting system targets a target site for TCF7. In some embodiments, the target site comprises the sequence set forth in any of SEQ ID NOs: 93- 98, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in any of SEQ ID NOs: 93-98, or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in any of SEQ ID NOs: 93-98. In some embodiments, the DNA-targeting system targets a target site for TCF7 comprising the sequence set forth in SEQ ID NO:93. In some embodiments, the DNA-targeting system targets a target site for TCF7 comprising the sequence set forth in SEQ ID NO:94. In some embodiments, the DNA-targeting system targets a target site for TCF7 comprising the sequence set forth in SEQ ID NO:95. In some embodiments, the DNA-targeting system targets a target site for TCF7 comprising the sequence set forth in SEQ ID NO:96. In some embodiments, the DNA-targeting system targets a target site for TCF7 comprising the sequence set forth in SEQ ID NO:97. In some embodiments, the DNA-targeting system targets a target site for TCF7 comprising the sequence set forth in SEQ ID NO:98.

[0184] In some embodiments, the DNA-targeting system targets a target site for GATA3. In some embodiments, the target site comprises the sequence set forth in any of SEQ ID NOs: 99-104, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in any of SEQ ID NOs: 99-104, or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in any of SEQ ID NOs: 99-104. In some embodiments, the DNA-targeting system targets a target site for GATA3 comprising the sequence set forth in SEQ ID NO:99. In some embodiments, the DNA-targeting system targets a target site for GATA3 comprising the sequence set forth in SEQ ID NO: 100. In some embodiments, the DNA-targeting system targets a target site for GATA3 comprising the sequence set forth in SEQ ID NO: 101. In some embodiments, the DNA-targeting system targets a target site for GATA3 comprising the sequence set forth in SEQ ID NO: 102. In some embodiments, the DNA-targeting system targets a target site for GATA3 comprising the sequence set forth in SEQ ID NO: 103. In some embodiments, the DNA-targeting system targets a target site for GATA3 comprising the sequence set forth in SEQ ID NO: 104.

[0185] In some embodiments, the DNA-targeting system targets a target site for BCL1 IB. In some embodiments, the target site comprises the sequence set forth in any of SEQ ID NOs: 105-110, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in any of SEQ ID NOs: 105-110, or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in any of SEQ ID NOs: 105-110. In some embodiments, the DNA-targeting system targets a target site for BCL1 IB comprising the sequence set forth in SEQ ID NO: 105. In some embodiments, the DNA-targeting system targets a target site for BCL1 IB comprising the sequence set forth in SEQ ID NO: 106. In some embodiments, the DNA-targeting system targets a target site for BCL1 IB comprising the sequence set forth in SEQ ID NO: 107. In some embodiments, the DNA-targeting system targets a target site for BCL1 IB comprising the sequence set forth in SEQ ID NO: 108. In some embodiments, the DNA-targeting system targets a target site for BCL11B comprising the sequence set forth in SEQ ID NO: 109. In some embodiments, the DNA-targeting system targets a target site for BCL1 IB comprising the sequence set forth in SEQ ID NO: 110.

[0186] In some embodiments, the DNA-targeting system targets a target site for TCF7 comprising the sequence set forth in SEQ ID NO:93. In some embodiments, the DNA-targeting system targets a target site for GATA3 comprising the sequence set forth in SEQ ID NO: 103. In some embodiments, the DNA-targeting system targets a target site for BCL1 IB comprising the sequence set forth in SEQ ID NO: 108. In some embodiments, the DNA-targeting system targets a target site for TCF7 comprising the sequence set forth in SEQ ID NO:93, a target site for GATA3 comprising the sequence set forth in SEQ ID NO: 103, and a target site for BCL1 IB comprising the sequence set forth in SEQ ID NO: 108.

[0187] In some embodiments, the target site for RUNX3 is located within 550bp of human genome assembly GRCh38 (hg38) genomic coordinates chrl: 24,930,276. In some embodiments the target site for RUNX3 is located within the genomic coordinates human genome assembly GRCh38 (hg38) chrl: 24,930,911- 24,929,930. In some embodiments the target site for RUNX3 is located within the genomic coordinates human genome assembly GRCh38 (hg38) chrl: 24,930,661- 24,930,180. In some embodiments the target site for RUNX3 is located within the genomic coordinates human genome assembly GRCh38 (hg38) chrl: 24,930,511- 24,930,330. In some embodiments the target site for RUNX3 is located within the genomic coordinates human genome assembly GRCh38 (hg38) chrl: 24,930,461- 24,930,380.

[0188] In some embodiments, the target site for IL7Ra is located within 550bp of human genome assembly GRCh38 (hg38) genomic coordinates chr5: 35,856,891. In some embodiments the target site for IL7Ra is located within the genomic coordinates human genome assembly GRCh38 (hg38) chr5: 35,857,258- 35,856,277. In some embodiments the target site for IL7Ra is located within the genomic coordinates human genome assembly GRCh38 (hg38) chr5: 35,857,008- 35,856,527. In some embodiments the target site for IL7Ra is located within the genomic coordinates human genome assembly GRCh38 (hg38) chr5: 35,856,858- 35,856,677. In some embodiments the target site for IL7Ra is located within the genomic coordinates human genome assembly GRCh38 (hg38) chr5: 35,856,808- 35,856,727.C. CRISPR / Cas-Based DNA-Targeting Systems and DNA-Binding Domains

[0189] In some embodiments, any of the provided DNA-targeting systems are based on CRISPR / Cas systems, i.e. CRISPR / Cas-based DNA-targeting systems, that are able to bind to a target site for a target gene as described, or to a combination of target sites, e.g. for a combination of target genes as described. In some embodiments, the CRISPR / Cas DNA- binding domain is nuclease inactive, such as includes a dCas (e.g. dCas9) so that the system binds to the target site for a target gene without mediating nucleic acid cleavage at the target site. The CRISPR / Cas-based DNA-targeting systems may be used to modulate expression of a target gene in a cell, such as a T cell. In some embodiments, the target gene may include any as described herein, including any described above in Section I.B. In some embodiments, the target site for the target gene may include any as described herein, including any described above in Section I.B. In some embodiments, the CRISPR / Cas-based DNA-targeting system can include any known Cas enzyme, and generally a nuclease-inactive or dCas. In some embodiments, the CRISPR / Cas-based DNA-targeting system includes a fusion protein of a nuclease-inactive Cas protein or a variant thereof and an effector domain, and at least one gRNA. In some embodiments, the effector domain increases transcription of the one or more genes (e.g. the effector domain is a transcriptional activator, such as any described in Section I.E).

[0190] The CRISPR system (also known as CRISPR / Cas system, or CRISPR-Cas system) refers to a conserved microbial nuclease system, found in the genomes of bacteria and archaea, that provides a form of acquired immunity against invading phages and plasmids. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), refers to loci containing multiple repeating DNA elements that are separated by non-repeating DNA sequences called spacers.Spacers are short sequences of foreign DNA that are incorporated into the genome between CRISPR repeats, serving as a “memory” of past exposures. Spacers encode the DNA-targeting portion of RNA molecules that confer specificity for nucleic acid cleavage by the CRISPR system. CRISPR loci contain or are adjacent to one or more CRISPR-associated (Cas) genes, which can act as RNA-guided nucleases for mediating the cleavage, as well as non-protein coding DNA elements that encode RNA molecules capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage.

[0191] In Type II CRISPR / Cas systems with the Cas protein Cas9, two RNA molecules and the Cas9 protein form a ribonucleoprotein (RNP) complex to direct Cas9 nuclease activity. The CRISPR RNA (crRNA) contains a spacer sequence that is complementary to a target nucleic acid sequence (target site), and that encodes the sequence specificity of the complex. The transactivating crRNA (tracrRNA) base-pairs to a portion of the crRNA and forms a structure that complexes with the Cas9 protein, forming a Cas / RNA RNP complex.

[0192] Naturally occurring CRISPR / Cas systems, such as those with Cas9, have been engineered to allow efficient programming of Cas / RNA RNPs to target desired sequences in cells of interest, both for gene-editing and modulation of gene expression. The tracrRNA and crRNA have been engineered to form a single chimeric guide RNA molecule, commonly referred to as a guide RNA (gRNA), for example as described in WO 2013 / 176772, WO 2014 / 093661, WO 2014 / 093655, Jinek, M. et al. Science 337(6096):816-21 (2012), or Cong, L. et al. Science 339(6121): 819-23 (2013). The spacer sequence of the gRNA can be chosen by a user to target the Cas / gRNA RNP complex to a desired locus, e.g. a desired target site in the target gene.

[0193] Cas proteins have also been engineered to be catalytically inactivated or nuclease inactive to allow targeting of Cas / gRNA RNPs without inducing cleavage at the target site. Mutations in Cas proteins can reduce or abolish nuclease activity of the Cas protein, rendering the Cas protein catalytically inactive. Cas proteins with reduced or abolished nuclease activity are referred to as deactivated Cas (dCas), or nuclease-inactive Cas (iCas) proteins, as referred to interchangeably herein. An exemplary deactivated Cas9 (dCas9) derived from 5. pyogenes contains silencing mutations of the RuvC and HNH nuclease domains (D10A and H840A), for example as described in WO 2013 / 176772, WO 2014 / 093661, Jinek, M. et al. Science 337(6096):816-21 (2012), and Qi, L. et al. Cell 152(5): 1173-83 (2013). Exemplary dCas variants derived from the Casl2 system (i.e. Cpfl) are described, for example in WO2017 / 189308 and Zetsche, B. et al. Cell 163(3):759-71 (2015). Conserved domains that mediate nucleic acid cleavage, such as RuvC and HNH endonuclease domains, are readily identifiable in Cas orthologues, and can be mutated to produce inactive variants, for example as described in Zetsche, B. et al. Cell 163(3):759-71 (2015).

[0194] dCas-fusion proteins with transcriptional and / or epigenetic regulators have been used as a versatile platform for ectopically regulating gene expression in target cells. These include fusion of a Cas with an effector domain, such as a transcriptional activator or transcriptional repressor. For example, fusing dCas9 with a transcriptional activator such as VP64 (a polypeptide composed of four tandem copies of VP 16, a 16 amino acid transactivation domain of the Herpes simplex virus) can result in robust induction of gene expression. Alternatively, fusing dCas9 with a transcriptional repressor such as KRAB (Kruppel associated box) can result in robust repression of gene expression. A variety of dCas-fusion proteins with effector domains can be engineered for regulation of gene expression, for example as described in WO 2014 / 197748, WO 2016 / 130600, WO 2017 / 180915, WO 2021 / 226555, WO 2013 / 176772, WO 2014 / 152432, WO 2014 / 093661, WO 2021 / 247570, Adli, M. Nat. Commun. 9, 1911 (2018), Perez-Pinera, P. et al. Nat. Methods 10, 973-976 (2013), Mali, P. et al. Nat. Biotechnol. 31, 833-838 (2013), Maeder, M. L. et al. Nat. Methods 10, 977-979 (2013), Gilbert, L. A. et al. Cell 154(2):442-451 (2013), and Nunez, J.K. et al. Cell 184(9):2503-2519 (2021).

[0195] In some aspects, provided is a DNA-targeting system comprising a fusion protein comprising a DNA-binding domain comprising a nuclease-inactive Cas protein or variant thereof, and an effector domain for increasing transcription or inducing transcriptional activation (i.e. a transcriptional activator) when targeted to a target gene in a cell (e.g. a T cell). In some embodiments, the dCas protein is any suitable dCas protein, such as any described in Section I.C. In some embodiments, the dCas protein is a dCas9 protein, such dSpCas9 or dSaCas9. In some embodiments, the at least one effector domain is any suitable transcriptional activator effector domain, such as any described in Section I.E., such as VP64. In some embodiments, the at least one effector domain is VP64. In some embodiments, the fusion protein is a dCas9-VP64 fusion protein, for example as described in Section I.F. In such embodiments, the DNA-targeting system also includes one or more gRNAs (e.g. as described in Section I.C.2.), provided in combination or as a complex with the dCas protein or variant thereof, for targeting of the DNA- targeting system to the target site of the target gene. In some embodiments, the fusion protein is guided to a specific target site sequence of the target gene by the guide RNA, wherein theeffector domain mediates targeted epigenetic modification to increase or activate transcription of the target gene. In some embodiments, a combination of gRNAs guides the fusion protein to a combination of target site sequences in a combination of genes, wherein the effector domain mediates targeted epigenetic modification to increase or activate transcription of the combination of target genes. Any of a variety of effector domains that increase or activate transcription can be used as described further below. / . CP / SPP / Cas-Dased DNA-Dinding Domains

[0196] In some aspects, the DNA-binding domain comprises a CRISPR-associated (Cas) protein or variant thereof, or is derived from a Cas protein or variant thereof. In particular embodiments here, the Cas protein is nuclease-inactive (i.e. is a dCas protein).

[0197] In some embodiments, the Cas protein is derived from a Class 1 CRISPR system (i.e. multiple Cas protein system), such as a Type I, Type III, or Type IV CRISPR system. In some embodiments, the Cas protein is derived from a Class 2 CRISPR system (i.e. single Cas protein system), such as a Type II, Type V, or Type VI CRISPR system. In some embodiments, the Cas protein is from a Type V CRISPR system. In some embodiments, the Cas protein is derived from a Cas 12 protein (i.e. Cpfl) or variant thereof, for example as described in WO 2017 / 189308 and Zetsche, B. et al. Cell. 163(3):759-71 (2015). In some embodiments, the Cas protein is derived from a Type II CRISPR system. In some embodiments, the Cas protein is derived from a Cas9 protein or variant thereof, for example as described in WO 2013 / 176772, WO 2014 / 152432, WO 2014 / 093661, WO 2014 / 093655, Jinek, M. et al. Science 337(6096):816-21 (2012), Mali, P. et al. Science 339(6121):823-6 (2013), Cong, L. et al. Science 339(6121):819-23 (2013), Perez-Pinera, P. et al. Nat. Methods 10, 973-976 (2013), or Mali, P. et al. Nat. Biotechnol. 31, 833-838 (2013). Various CRISPR / Cas systems and associated Cas proteins for use in gene editing and regulation have been described, for example in Moon, S.B. et al. Exp. Mol. Med. 51, 1-11 (2019), Zhang, F. Q. Rev. Biophys. 52, E6 (2019), and Makarova K.S. et al. Methods Mol. Biol. 1311:47-75 (2015).

[0198] In some embodiments, the dCas9 protein can comprise a sequence derived from a naturally occurring Cas9 molecule, or variant thereof. In some embodiments, the dCas9 protein can comprise a sequence derived from a naturally occurring Cas9 molecule of .S'. pyogenes, S. thermophilus, S. aureus, C. jejuni, N. meningitidis, F. novicida, S. canis, S. auricularis, or variant thereof. In some embodiments, the dCas9 protein comprises a sequence derived from anaturally occurring Cas9 molecule of S. aureus. In some embodiments, the dCas9 protein comprises a sequence derived from a naturally occurring Cas9 molecule of S. pyogenes.

[0199] Non-limiting examples of Cas9 orthologs from other bacterial strains include but are not limited to: Cas proteins identified in Acaryochloris marina MBIC 11017; Acetohalobium arabaticum DSM 5501; Acidithiobacillus caldus; Acidithiobacillus ferrooxidans ATCC 23270; Alicyclobacillus acidocaldarius LAA1; Alicyclobacillus acidocaldarius subsp. Acidocaldarius DSM 446; Allochromatium vinosum DSM 180; Ammonifex degensii KC4; Anabaena variabilis ATCC 29413; Arthrospira maxima CS-328; Arthrospira platensis str. Paraca; Arthrospira sp. PCC 8005; Bacillus pseudomycoides DSM 12442; Bacillus selenitireducens MLS 10; Burkholderiales bacterium 1_1_47; Caldicelulosiruptor becscii DSM 6725; Candidatus Desulforudis audaxviator MP104C; Caldicellulosiruptor hydrothermalis 108; Clostridium phage c-st; Clostridium botulinum A3 str. Loch Maree; Clostridium botulinum Ba4 str. 657; Clostridium difficile QCD-63q42; Crocosphaera watsonii WH 8501; Cyanothece sp. ATCC 51142; Cyanothece sp. CCY0110; Cyanothece sp. PCC 7424; Cyanothece sp. PCC 7822; Exiguobacterium sibiricum 255-15; Finegoldia magna ATCC 29328; Ktedonobacter racemifer DSM 44963; Lactobacillus delbrueckii subsp. Bulgaricus PB2003 / 044-T3-4; Lactobacillus salivarius ATCC 11741; Listeria innocua; 64yngby asp. PCC 8106; Marinobacter sp. ELB 17; Methanohalobium evestigatum Z-7303; Microcystis phage Ma-LMMOl; Microcystis aeruginosa NIES-843; Microscilla marina ATCC 23134; Microcoleus chthonoplastes PCC 7420; Neisseria meningitidis; Nitrosococcus halophilus Nc4; Nocardiopsis dassonvillei subsp. Dassonvillei DSM 43111; Nodularia spumigena CCY9414; Nostoc sp. PCC 7120; Oscillatoria sp. PCC 6506; Pelotomaculum_thermopropionicum SI; Petrotoga mobilis SJ95; Polaromonas naphthalenivorans CJ2; Polaromonas sp. JS666; Pseudoalteromonas haloplanktis TAC125; Streptomyces pristinaespiralis ATCC 25486; Streptomyces pristinaespiralis ATCC 25486; Streptococcus thermophilus; Streptomyces viridochromogenes DSM 40736; Streptosporangium roseum DSM 43021; Synechococcus sp. PCC 7335; and Thermosipho africanus TCF52B (Chylinski et al., RNA Biol., 2013; 10(5): 726-737).

[0200] In some aspects, the Cas protein is a variant that lacks nuclease activity (i.e. is a dCas protein). In some embodiments, the Cas protein is mutated so that nuclease activity is reduced or eliminated. Such Cas proteins are referred to as deactivated Cas or dead Cas (dCas) or nucleaseinactive Cas (iCas) proteins, as referred to interchangeably herein. In some embodiments, thevariant Cas protein is a variant Cas9 protein that lacks nuclease activity or that is a deactivated Cas9 (dCas9, or iCas9) protein.

[0201] In some embodiments, the Cas9 protein or a variant thereof is derived from a Staphylococcus aureus Cas9 (SaCas9) protein or a variant thereof. In some embodiments, the variant Cas9 is a Staphylococcus aureus dCas9 protein (dSaCas9) that comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO:88. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO:89 or SEQ ID NO: 261, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0202] In some embodiments, the Cas9 protein or variant thereof is derived from a Streptococcus pyogenes Cas9 (SpCas9) protein or a variant thereof. In some embodiments, the variant Cas9 is a Streptococcus pyogenes dCas9 (dSpCas9) protein that comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO: 31. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO: 32 or SEQ ID NO: 259 , or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.2. Guide RlVAs (g GVAs)

[0203] In some embodiments, the Cas protein (e.g. dCas9) is provided in combination or as a complex with one or more guide RNA (gRNA). In some embodiments, the gRNA is capable of complexing with the Cas protein or variant thereof. In some embodiments, the gRNA comprises a gRNA spacer sequence (also known as a spacer sequence or a guide sequence) that is capable of hybridizing to the target site or is complementary to the target site, such as any target site described herein, for example, any target site in a genome. In some embodiments, the gRNA comprises a scaffold sequence that complexes with or binds to the Cas protein. In some embodiments, a gRNA specific to a target locus of interest (e.g. a regulatory DNA element of a DLL4 gene) is used to recruit an RNA-guided protein (e.g. a Cas protein) or variant thereof or a fusion protein comprising such RNA-guided protein (e.g., a Cas polypeptide), to the target site. In some aspects, the gRNA is a nucleic acid that promotes the specific targeting or homing of the gRNA / Cas RNP complex to the target site of the target gene, such as any described above in Section I.B. In some embodiments, a target site of a gRNA may be referred to as a protospacer.

[0204] Provided herein are gRNAs, such as gRNAs that target or bind to a target site for a gene, such as in a target gene or regulatory DNA element thereof, such as any described herein,for example in Section LB. Provided herein are gRNAs, such as gRNAs that target or can bind to a regulatory DNA element of a DLL4 gene. In some embodiments, the gRNAs bind to a target site that is located in the DLL4 gene and / or a regulatory DNA element of the DLL4 gene. In some embodiments, the gRNAs bind to a target site that is located in the DLL4 gene. In some embodiments, the gRNAs bind to a target site that is located in a regulatory DNA element of the LCD gene. Provided herein are gRNAs, such as gRNAs that target or can bind to a regulatory DNA element of a VCAM1 gene. In some embodiments, the gRNAs bind to a target site that is located in the VCAM1 gene and / or a regulatory DNA element of the VCAM1 gene. In some embodiments, the gRNAs bind to a target site that is located in the VCAM1 gene. In some embodiments, the gRNAs bind to a target site that is located in a regulatory DNA element of the VCAM1 gene. In some embodiments, gRNAs are provided that target or can bind to a regulatory DNA element of a LCD gene. In some embodiments, the gRNAs bind to a target site that is located in the LCD gene and / or a regulatory DNA element of the LCD gene. In some embodiments, the gRNAs bind to a target site that is located in the LCD gene. In some embodiments, the gRNAs bind to a target site that is located in a regulatory DNA element of the LCD gene.

[0205] In some embodiments, the gRNA is capable of complexing with the Cas protein or variant thereof. In some embodiments, any of the provided gRNA sequences is complexed with or is provided in combination with a fusion protein comprising Cas9. In some embodiments, the Cas9 is a dCas9. In some embodiments, the dCas9 is a dSpCas9, such as a dSpCas9. The Cas9 can be any as described herein such as in Section I.C.l. In some embodiments, the gRNA comprises a gRNA spacer sequence (i.e. a spacer sequence or a guide sequence) that is capable of hybridizing to the target site, or that is complementary to the target site, such as any target site described herein. In some embodiments, the gRNA comprises a scaffold sequence that complexes with or binds to the Cas protein.

[0206] In some aspects, a “gRNA molecule” is a nucleic acid that promotes the specific targeting or homing of a gRNA molecule / Cas9 molecule complex to a target nucleic acid, such as a locus on the genomic DNA of a cell. In general, a spacer sequence of the guide RNA, is any polynucleotide sequences comprising at least a sequence portion that has sufficient complementarity with a target polynucleotide sequence, such as the at a DLL4 gene locus in humans, to hybridize with the target sequence at the target site and direct sequence- specific binding of the CRISPR complex to the target sequence. In some embodiments, in the context offormation of a CRISPR complex, “target sequence” is to a sequence to which a spacer sequence is designed to have complementarity, where hybridization between the target sequence and a spacer sequence of the guide RNA promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. Generally, a spacer sequence is selected to reduce the degree of secondary structure within the spacer sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm.

[0207] In some embodiments, a guide RNA (gRNA) specific to a target locus of interest (e.g. at a DLL4 gene locus) is used with RNA-guided nucleases or variants thereof, e.g., nuclease-inactive Cas variants, to target the provided DNA-targeting system to the target site or target position. Methods for designing gRNAs and exemplary spacer sequences are known. Exemplary gRNA structures that can be associated with particular RNA-guided nucleases or variants thereof, e.g., nuclease-inactive Cas variants, with particular domains and scaffold regions, are also known. In some aspects, gRNA molecules comprise a scaffold sequence, e.g., sequences that can be complexed with the Cas protein.

[0208] In some embodiments, the scaffold is specific for a Cas protein. In some embodiments, the scaffold is specific for SpCas9 or a dSpCas9. In some embodiments the scaffold comprises the nucleic acid sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 23. In some embodiments the scaffold comprises the nucleic acid sequence set forth in SEQ ID NO: 82 or SEQ ID NO: 83. In some embodiments, the scaffold is specific for a SaCas9 or a dSaCas9. In some embodiments, the scaffold comprises the nucleic acid sequence set forth in SEQ ID NO: 262 or SEQ ID NO: 263.

[0209] In some embodiments, the gRNA comprises a scaffold sequence comprising the nucleic acid sequence set forth in SEQ ID NO: 23 and any one of the gRNA spacer sequences provided herein. In some embodiments, the gRNA comprises a scaffold sequence comprising the nucleic acid sequence set forth in SEQ ID NO: 23 and a spacer sequence comprising the sequence selected from any one of SEQ ID NOs: 8-14, 54-67, 111-128, and 167-186. In some embodiments, the gRNA comprises the sequence selected from any one of SEQ ID NOs: 15-21, 68-81, and 129-146.

[0210] In some embodiments, the gRNA comprises a scaffold sequence comprising the nucleic acid sequence set forth in SEQ ID NO: 83 and any one of the gRNA spacer sequences provided herein. In some embodiments, the gRNA comprises a scaffold sequence comprisingthe nucleic acid sequence set forth in SEQ ID NO: 83 and a spacer sequence comprising the sequence selected from any one of SEQ ID NOs: 8-14, 54-67, 111-128, and 167-186. In some embodiments, the gRNA comprises the sequence selected from any one of SEQ ID NOs: 33-39.

[0211] In some embodiments, the gRNA comprises a scaffold sequence comprising the nucleic acid sequence set forth in SEQ ID NO: 263 and any one of the gRNA spacer sequences provided herein. In some embodiments, the gRNA comprises a scaffold sequence comprising the nucleic acid sequence set forth in SEQ ID NO: 263 and a spacer sequence comprising the sequence selected from any one of SEQ ID NOs: 8-14, 54-67, 111-128, and 167-186.

[0212] A guide RNA can comprise at least a spacer sequence that hybridizes to a target nucleic acid sequence of interest, and a CRISPR repeat sequence. In Type II systems, the gRNA also comprises a second RNA called the tracrRNA sequence. In the Type II guide RNA (gRNA), the CRISPR repeat sequence and tracrRNA sequence hybridize to each other to form a duplex. In the Type V guide RNA (gRNA), the crRNA forms a duplex. In both systems, the duplex can bind a site-directed polypeptide, such that the guide RNA and site-direct polypeptide form a complex. The gRNA can provide target specificity to the complex by virtue of its association with the site-directed polypeptide. The gRNA thus can direct the activity of the site- directed polypeptide.

[0213] In some embodiments, the gRNAs provided herein are chimeric gRNAs. In general, gRNAs can be unimolecular (i.e. composed of a single RNA molecule), or modular (comprising more than one, and typically two, separate RNA molecules). Modular gRNAs can be engineered to be unimolecular, wherein sequences from the separate modular RNA molecules are comprised in a single gRNA molecule, sometimes referred to as a chimeric gRNA, synthetic gRNA, or single gRNA. In some embodiments, the chimeric gRNA is a fusion of two noncoding RNA sequences: a crRNA sequence and a tracrRNA sequence, for example as described in WO 2013 / 176772, or Jinek, M. et al. Science 337(6096):816-21 (2012). In some embodiments, the chimeric gRNA mimics the naturally occurring crRNA:tracrRNA duplex involved in the Type II Effector system, wherein the naturally occurring crRNA:tracrRNA duplex acts as a guide for the Cas9 protein.

[0214] In some embodiments the gRNA is a concatenation of two non-coding RNA sequences: a crRNA sequence and a tracrRNA sequence. The gRNA may target a desired DNA sequence by exchanging the sequence encoding a 20 bp protospacer which confers targeting specificity through complementary base pairing with the desired DNA target. gRNA mimics thenaturally occurring crRNA:tracrRNA duplex involved in the Type II CRISPR / Cas system (e.g., Cas9). This duplex, which may include, for example, a 42-nucleotide crRNA and a 75- nucleotide tracrRNA, acts as a guide for the Cas9 protein to cleave the target nucleic acid. The CRISPR / Cas9-based system may include two or more gRNAs, wherein the two or more gRNAs target different DNA sequences. The target DNA sequences may be overlapping or nonoverlapping. The target DNA sequences may be located within or near the same gene or different genes. The target sequence or protospacer is followed by a PAM sequence at the 3' end of the protospacer. Different Type II systems have differing PAM requirements. For example, the Streptococcus pyogenes Type II system uses an “NGG” sequence 3’ (SEQ ID NO: 90), where “N” can be any nucleotide.

[0215] In some aspects, the spacer sequence of a gRNA is a polynucleotide sequence comprising at least a portion that has sufficient complementarity with the target site to hybridize with the target site in the target gene and direct sequence- specific binding of a Cas / gRNA complex to the sequence of the target site. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization. In some embodiments, the gRNA comprises a spacer sequence that is complementary, e.g., at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% (e.g., fully complementary), to the target site. The strand of the target nucleic acid comprising the target site sequence may be referred to as the “complementary strand” of the target nucleic acid.

[0216] In some aspects, a gRNA targets a target site in double- stranded DNA. Thus, in some aspects, the sequence of the target site may be defined by the sequence that the gRNA spacer hybridizes to, or by the sequence complementary to the sequence that the gRNA spacer hybridizes to. In some aspects, the sequence of the target site may be defined by the sequence that the gRNA spacer displaces in order to hybridize to the DNA. In some embodiments, the sequence of the target site is the sequence that the gRNA hybridizes to.

[0217] In some embodiments, the gRNA spacer sequence is between about 14 nucleotides (nt) and about 26 nt, or between 16 nt and 22 nt in length. In some embodiments, the gRNA spacer sequence is 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt or 22 nt, 23 nt, 24 nt, 25 nt, or 26 nt in length. In some embodiments, the gRNA spacer sequence is 18 nt, 19 nt, 20 nt, 21 nt or 22 nt in length. In some embodiments, the gRNA spacer sequence is 20 nt in length.

[0218] A target site of a gRNA may be referred to as a protospacer. In some aspects, the spacer is designed to target a protospacer with a specific protospacer-adjacent motif (PAM), i.e.a sequence immediately adjacent to the protospacer that contributes to and / or is required for Cas binding specificity. Different CRISPR / Cas systems have different PAM requirements for targeting. For example, in some embodiments, .S'. pyogenes Cas9 uses the PAM 5’-NGG-3’ (SEQ ID NO: 90), where N is any nucleotide. In some embodiments, the PAM of a gRNA for complexing with a Type V CRISPR / Cas system, such as with Casl2a (also known as Cpfl) or variant thereof uses TTTV (SEQ ID NO: 242), where V is A, C, or G. In some embodiments, .S'. aureus Cas9 uses the PAM 5’- NNGRRT-3’ (SEQ ID NO: 91), where N is any nucleotide, and R is G or A. In some embodiments, N. meningitidis Cas9 uses the PAM 5'-NNNNGATT-3’ (SEQ ID NO: 243), where N is any nucleotide. In some embodiments, C. jejuni Cas9 uses the PAM 5'-NNNNRYAC-3' (SEQ ID NO: 244), where N is any nucleotide, R is G or A, and Y is C or T. In some embodiments, .S. thermophilus uses the PAM 5’-NNAGAAW-3’ (SEQ ID NO: 245), where N is any nucleotide and W is A or T. In some embodiments, F. Novicida Cas9 uses the PAM 5’-NGG-3’ (SEQ ID NO: 90), where N is any nucleotide. In some embodiments, T. denticola Cas9 uses the PAM 5’-NAAAAC-3’ (SEQ ID NO: 246), where N is any nucleotide.In some embodiments, Cas proteins may use or be engineered to use different PAMs from those listed above. For example, mutated SpCas9 proteins may use the PAMs 5’-NGG-3’ (SEQ ID NO:90), 5’-NGAN-3’ (SEQ ID NO: 247), 5’-NGNG-3’ (SEQ ID NO: 248), 5’-NGAG-3’ (SEQ ID NO: 218), or 5’-NGCG-3’ (SEQ ID NO: 249), where N is any nucleotide. In some embodiments, the protospacer-adjacent motif (PAM) of a gRNA for complexing with .S’. pyogenes Cas9 or variant thereof is NGG, as set forth in SEQ ID NO: 90. In some embodiments, the PAM of a gRNA for complexing with .S’, aureus Cas9 or variant thereof is NNGRRT, as set forth in SEQ ID NO: 91. Methods for designing or identifying gRNA spacer sequences and / or protospacer sequences in a particular region, are known. gRNA spacer sequences and / or protospacer sequences can be determined based on the type of Cas protein used and the associated PAM sequence.

[0219] A spacer sequence may be selected to reduce the degree of secondary structure within the spacer sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm.

[0220] In some embodiments, the gRNA (including the guide sequence) will comprise the base uracil (U), whereas DNA encoding the gRNA molecule will comprise the base thymine (T). While not wishing to be bound by theory, in some embodiments, it is believed that the complementarity of the guide sequence with the target sequence contributes to specificity of theinteraction of the gRNA molecule / Cas molecule complex with a target nucleic acid. It is understood that in a guide sequence and target sequence pair, the uracil bases in the guide sequence will pair with the adenine bases in the target sequence.

[0221] In some embodiments, the gRNA comprises modified nucleotides, e.g. for increased stability. In some embodiments, one, more than one, or all of the nucleotides of a gRNA can have a modification, e.g., to render the gRNA less susceptible to degradation and / or improve bio-compatibility. By way of non-limiting example, the backbone of the gRNA can be modified with a phosphorothioate, or other modification(s). In some cases, a nucleotide of the gRNA can comprise a 2’ modification, e.g., a 2-acetylation, e.g., a 2’ methylation, or other modification(s).

[0222] Methods for designing gRNAs and exemplary targeting domains can include those described in, e.g., International PCT Pub. Nos. WO 2014 / 197748, WO 2016 / 130600, WO 2017 / 180915, WO 2021 / 226555, WO 2013 / 176772, WO 2014 / 152432, WO 2014 / 093661, WO 2014 / 093655, WO 2015 / 089427, WO 2016 / 049258, WO 2016 / 123578, WO 2021 / 076744, WO 2014 / 191128, WO 2015 / 161276, WO 2017 / 193107, and WO 2017 / 093969.

[0223] In some aspects, the gRNA comprises scaffold sequences. In some aspects, the scaffold sequence (in some cases including a crRNA sequence and / or a tracrRNA sequence) will be different depending on the Cas protein. In some aspects, different CRISPR / Cas systems have different gRNA scaffold sequences for associating with Cas protein. In some embodiments, an exemplary scaffold sequence for .S'. aureus Cas9 comprises a sequence set forth in SEQ ID NO: 263, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 263. In some embodiments, an exemplary scaffold sequence for .S'. aureus Cas9 comprises a sequence set forth in SEQ ID NO: 263.

[0224] In some embodiments, an exemplary scaffold sequence for .S'. pyogenes Cas9 comprises a sequence set forth in SEQ ID NO:23, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:56. In some embodiments, an exemplary scaffold sequence for .S'. pyogenes Cas9 comprises a sequence set forth in SEQ ID NO: 23. In some embodiments, an exemplary scaffold sequence for .S', pyogenes Cas9 comprises a sequence set forth in SEQ ID NO:83, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:56. In someembodiments, an exemplary scaffold sequence for .S'. pyogenes Cas9 comprises a sequence set forth in SEQ ID NO: 83.

[0225] In some embodiments, an exemplary scaffold sequence for Acidaminococcus sp. Casl2a comprises a sequence set forth in SEQ ID NO:250, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 250. In some embodiments, an exemplary scaffold sequence for CasPhi-2 comprises a sequence set forth in SEQ ID NO: 251, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 251. In some embodiments, an exemplary scaffold sequence for UnlCasl2fl comprises a sequence set forth in SEQ ID NO: 252 , the sequence “TTTTATTTT” (SEQ ID NO: 253), the sequence “GGAATGAAC” (SEQ ID NO: 254), or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:252, 253 or 254. In some embodiments, an exemplary scaffold sequence for UnlCasl2fl comprises a sequence set forth in SEQ ID NO:252 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:252. In some embodiments, an exemplary scaffold sequence for UnlCasl2fl comprises a sequence set forth in SEQ ID NO:253, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:253. In some embodiments, an exemplary scaffold sequence for UnlCasl2fl comprises a sequence set forth in SEQ ID NO:254, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:254. In some embodiments, an exemplary scaffold sequence for C. jejuni Cas9 comprises a sequence set forth in SEQ ID NO:255, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:255. In some embodiments, an exemplary scaffold sequence for Casl2k comprises a sequence set forth in SEQ ID NO:256, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:256. In some embodiments, an exemplary scaffold sequence for CasMini comprises a sequence set forth in SEQ ID NO:257, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:257.

[0226] In some aspects, the gRNA can target the DNA-targeting system to direct the activities of an associated polypeptide (e.g., fusion protein, DNA-targeting system, effector domain, etc.) to a specific target site within a target nucleic acid (e.g., regulatory DNA element of a DLL4 gene locus).

[0227] In some embodiments, a gRNA provided herein targets a target site for a DLL4 gene or regulatory element thereof for transcriptional activation. In some embodiments, a gRNA provided herein targets a target site for a DLL4 gene or regulatory element thereof for transcriptional activation. In some embodiments, the target site is located on a DLL4 gene. In some embodiments, the target site is located in a regulatory DNA element of a DLL4 gene. In some embodiments, a regulatory DNA element is a sequence to which a gene regulatory protein may bind and affect transcription of a DLL4. In some embodiments, a regulatory DNA element is a sequence to which a gene regulatory protein may bind and affect transcription of a DLL4 gene.

[0228] In some embodiments, a gRNA provided herein targets a target site for a VCAM1 gene or regulatory element thereof for transcriptional activation. In some embodiments, a gRNA provided herein targets a target site for a VC AMI gene or regulatory element thereof for transcriptional activation. In some embodiments, the target site is located on a VCAM1 gene. In some embodiments, the target site is located in a regulatory DNA element of a VCAMlgene. In some embodiments, a regulatory DNA element is a sequence to which a gene regulatory protein may bind and affect transcription of a VCAM1. In some embodiments, a regulatory DNA element is a sequence to which a gene regulatory protein may bind and affect transcription of a VC AMI gene.

[0229] In some embodiments, a gRNA provided herein targets a target site for an LCD gene or regulatory element thereof for transcriptional activation. In some embodiments, a gRNA provided herein targets a target site for an LCD gene or regulatory element thereof for transcriptional activation. In some embodiments, the target site is located on an LCD gene. In some embodiments, the target site is located in a regulatory DNA element of an LCD gene. In some embodiments, a regulatory DNA element is a sequence to which a gene regulatory protein may bind and affect transcription of an LCD gene. In some embodiments, a regulatory DNA element is a sequence to which a gene regulatory protein may bind and affect transcription of an LCD gene.

[0230] Exemplary target sites and combinations of target sites for gRNAs of the DNA-binding systems, including multiplexed DNA-binding systems, include any described in SectionLB.

[0231] In some embodiments, a gRNA provided herein targets a target site for a gene for transcriptional activation, such as any target site or gene described in Section LB. In some embodiments, a gRNA provided herein targets a target site for DLL4. In some embodiments, a gRNA provided herein targets a target site for VCAM1. In some embodiments, a gRNA provided herein targets a target site for an LCD gene, wherein the gene is selected from: MYB, RUNX3, RUNX1, SPI1, HEY1, IL7Ra, TBX21, LEF1, CBFB, TCF7, GATA3, and BCL11B.

[0232] In some embodiments, the gRNA targets a target site of the DLL4 gene that comprises a sequence selected from any one of SEQ ID NOS: 1-2 and 147-166, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site for DLL4 is a contiguous portion of any one of SEQ ID NOS: 1-2 and 147-166 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site for DLL4 is set forth in any one of SEQ ID NOS: 1-2 and 147-166. In some embodiments, the target site for DLL4 is set forth in SEQ ID NO: 2. In some embodiments, the target site for DLL4 is set forth in SEQ ID NO: 154.

[0233] In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:1, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 1, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 1 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:1. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:2, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 2, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 2 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:2.

[0234] In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 147, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 147, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 147 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 147. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 148, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 148, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 148. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 148 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 148. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 149, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 149, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 149. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 149 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 149. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 150, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 150, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 150. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 150 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 150. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 151, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 151, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 151. In some embodiments, the target site is a contiguous portion ofSEQ ID NO: 151 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 151. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 152, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 152, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 152. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 152 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 152. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 153, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 153, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 153. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 153 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 153. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 154, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 154, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 154. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 154 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 154. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 155, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 155, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 155. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 155 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 155. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 156, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 156, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 156 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 156. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 157, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 157, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 157. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 157 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 157. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 158, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 158, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 158. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 158 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 158. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 159, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 159, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 159. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 159 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 159. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 160, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 161, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 161. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 161 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 161. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 162, a contiguous portionthereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 162, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 162. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 162 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 162. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 163, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 163, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 163. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 163 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 163. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 164, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 164, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 164. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 164 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 164. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 165, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 165, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 165. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 165 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 165. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 166, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 166, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 166. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 166 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence setforth in SEQ ID NO:166.

[0235] In some embodiments, the gRNA comprises a spacer sequence for DLL4 selected from any one of SEQ ID NOS:8-9 and 167-186 or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of any one of SEQ ID NOS:8-9 and 167-186 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in any one of SEQ ID NOS: 8-9 and 167-186. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 9. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 174.

[0236] In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 8, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:8 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:8. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 9, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 9 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 9. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 167, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 167 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 167. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 168, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguousportion of SEQ ID NO: 168 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 168. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 169, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 169 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 169. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 170, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 170 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 170. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 171, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 171 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 171. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 172, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 172 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 172. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 173, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 173 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 173. In someembodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 174, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 174 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 174. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 175, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 175 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 175. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 176, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 176 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 176. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 177, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 177 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 177. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 178, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 178 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 178. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 179, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 179 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 179. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 180, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 180 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 180. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 181, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 181 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 181. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 182, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 182 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 182. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 183, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 183 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 183. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 184, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguousportion of SEQ ID NO: 184 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 184. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 185, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 185 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 185. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 186, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 186 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 186.

[0237] In some embodiments, the gRNA targeting a target site for DLL4 comprises a sequence comprising a spacer sequence set forth in any of SEQ ID NOs: 8, 9 and 167-186, and further comprises a scaffold sequence, such as any scaffold sequence disclosed herein. In some embodiments, the gRNA targeting a target site for DLL4comprises modified nucleotides.

[0238] In some embodiments, the gRNA comprises a sequence selected from any one of SEQ ID NOs: 15-16 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA is a contiguous portion of any one of SEQ ID NOs: 15-16. In some embodiments, the gRNA is set forth in any one of SEQ ID NOs: 15-16.

[0239] In some embodiments, the gRNA comprises modified nucleotides. In some embodiments, the gRNA comprises a sequence selected from any one of SEQ ID NOs: 33-34 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA is a contiguous portion of any one of SEQ ID NOs: 33-34. In some embodiments, the gRNA is set forth in any one of SEQ ID NOs: 33-34.

[0240] In some embodiments, the gRNA targets a target site of the VCAM1 gene that comprises a sequence selected from any one of SEQ ID NOS:3-7, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequencehaving at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site for VCAM1 is a contiguous portion of any one of SEQ ID NOS:3-7 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site for VCAM1 is set forth in any one of SEQ ID NOS: 3-7. In some embodiments, the target site for VCAM1 is set forth in SEQ ID NO: 3.

[0241] In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:3, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 3, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 3. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 3 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 3. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:4, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 4, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 4 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 4. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:5, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 5, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 5 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 5. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 6, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 6, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 6. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 6 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises thesequence set forth in SEQ ID NO: 6. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 7, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 7, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 7. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 7 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 7.

[0242] In some embodiments, the gRNA comprises a spacer sequence for VCAM1 selected from any one of SEQ ID NOS: 10- 14 or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of any one of SEQ ID NOS: 10- 14 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in any one of SEQ ID NOS: 10-14. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 10.

[0243] In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 10, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 10 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 10. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 11, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 11 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 11. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 12, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 12 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In someembodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 12. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 13, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 13 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 13. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 14, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 14 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 14.

[0244] In some embodiments, the gRNA targeting a target site for VCAM1 comprises a sequence comprising a spacer sequence set forth in any of SEQ ID NOs: 10-14, and further comprises a scaffold sequence, such as any scaffold sequence disclosed herein. In some embodiments, the gRNA targeting a target site for VCAM1 comprises modified nucleotides.

[0245] In some embodiments, the gRNA comprises a sequence selected from any one of SEQ ID NOs: 17-21 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA is a contiguous portion of any one of SEQ ID NOs: 17-21. In some embodiments, the gRNA is set forth in any one of SEQ ID NOs: 17-21.

[0246] In some embodiments, the gRNA comprises modified nucleotides. In some embodiments, the gRNA comprises a sequence selected from any one of SEQ ID NOs: 35-39 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA is a contiguous portion of any one of SEQ ID NOs: 35-39. In some embodiments, the gRNA is set forth in any one of SEQ ID NOs: 35-39.

[0247] In some embodiments, the gRNA targets a target site of an LCD gene that comprises a sequence selected from any one of SEQ ID NOS:40-53 and 93-110, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOS: 40-53 and SEQ ID NOS: 93- 110 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in any one of SEQ ID NOS: 40-53 and SEQ ID NOS: 93-110.

[0248] In some embodiments, the gRNA comprises a spacer sequence for an LCD gene selected from any one of SEQ ID NOS:54-67 and SEQ ID NOS: 111-128 or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of any one of SEQ ID NOS:54-67 and SEQ ID NOS: 111-128 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in any one of SEQ ID NOS:54-67 and SEQ ID NOS: 111-128.

[0249] In some embodiments, the gRNA comprises a sequence selected from any one of SEQ ID NOs: 68-81 and 129-146, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA is a contiguous portion of any one of SEQ ID NOs: 68-81 and 129-146. In some embodiments, the gRNA is set forth in any one of SEQ ID NOs: 68-81 and 129-146.

[0250] In some embodiments, the gRNA targets a target site for TCF7. In some embodiments, the gRNA targets the target site that comprises a sequence selected from any one of SEQ ID NOs: 93-98, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOs: 93-98 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in any one of SEQ ID NOs: 93-98. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 93, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 93, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 93. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 1 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence setforth in SEQ ID NO: 93. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 94, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 94, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 94. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 94 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 94. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 95, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 95, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 95. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 95 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:95. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:96, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 96, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 96. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 4 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:96. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:96, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 97, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 97. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 97 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:97. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:98, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 98, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 98. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 98 that is 14, 15, 16,17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:98.

[0251] In some embodiments, the gRNA targeting a target site for TCF7 comprises a spacer sequence selected from any one of SEQ ID NOs: 111-116, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of any one of SEQ ID NOs: 111-116that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in any one of SEQ ID NOS: 111-116. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 111, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 111. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 111. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 111. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 112, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 112. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 112. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:113, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 113. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 113. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 113. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 114, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 114. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 114. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 114. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 115, or a contiguous portion thereof of at least 14 nt, or a sequence having at orat least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 115. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 115. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 115. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:116, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 116. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 116. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 116.

[0252] In some embodiments, the gRNA targeting a target site for TCF7 comprises a sequence comprising a spacer sequence set forth in any of SEQ ID NOs: 111-116 and further comprises a scaffold sequence, such as any scaffold sequence disclosed herein. In some embodiments, the gRNA targeting a target site for TCF7 comprises modified nucleotides.

[0253] In some embodiments, the gRNA targets a target site for GATA3. In some embodiments, the gRNA targets the target site that comprises a sequence selected from any one of SEQ ID NOs: 99-104, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOs: 99-104, that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in any one of SEQ ID NOs: 99-104. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:99, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 99, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 99 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:99. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 100, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 100, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 100. In some embodiments, the target site is acontiguous portion of SEQ ID NO: 100 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 100. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 101, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 101, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 101. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 101 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 101. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 102, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 102, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 102. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 102 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 102. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 103, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 103, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 103. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 103 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 103. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 104, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 104, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 104. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 104 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 104.

[0254] In some embodiments, the gRNA targeting a target site for GATA3 comprises a spacer sequence selected from any one of SEQ ID NOs:117-122, or a contiguous portion thereofof at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of any one of SEQ ID NOs: 117-122that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in any one of SEQ ID NOS: 117- 122. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 117, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 117. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 117. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 117. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 118, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 118. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 118. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 118. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 119, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 119. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 119. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 119. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 120, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 120. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 120. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 120. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 121, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:121. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 121. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 121. In someembodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 122, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 122. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 122. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 122.

[0255] In some embodiments, the gRNA targeting a target site for GATA3 comprises a sequence comprising a spacer sequence set forth in any of SEQ ID NOs: 117-122 and further comprises a scaffold sequence, such as any scaffold sequence disclosed herein. In some embodiments, the gRNA targeting a target site for GATA3 comprises modified nucleotides.

[0256] In some embodiments, the gRNA targets a target site for BCL1 IB. In some embodiments, the gRNA targets the target site that comprises a sequence selected from any one of SEQ ID NOs: 105- 110, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOs: 105-110 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in any one of SEQ ID NOs: 105-110. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 105, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 105, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 105. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 105 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 105. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 106, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 106 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 106. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 106 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 106. In some embodiments, the gRNA targets the target site that comprisesthe sequence set forth in SEQ ID NO: 107, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 107, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 107. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 107 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 107. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 108, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 108, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 108. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 108 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 108. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 109, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 109, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 109. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 109 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 109. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 110, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 110, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 110. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 110 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 110.

[0257] In some embodiments, the gRNA targeting a target site for BCL1 IB comprises a spacer sequence selected from any one of SEQ ID NOs:123-128, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of any one ofSEQ ID NOs: 123-128 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in any one of SEQ ID NOS: 123- 128. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 123, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 123. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 123. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 124, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 124. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 124. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 124. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 125, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 125. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 125. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 125. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 126, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 126. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 126. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 126. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 127, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 127. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 127. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 127. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 128, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identityto SEQ ID NO: 128. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 128. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 128.

[0258] In some embodiments, the gRNA targeting a target site for BCL1 IB comprises a sequence comprising a spacer sequence set forth in any of SEQ ID NOs: 123-128 and further comprises a scaffold sequence, such as any scaffold sequence disclosed herein. In some embodiments, the gRNA targeting a target site for BCL11B comprises modified nucleotides.

[0259] In some embodiments, the gRNA targets a target site for MYB. In some embodiments, the gRNA targets the target site that comprises a sequence set forth in SEQ ID NO:40 or SEQ ID NO:41, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of SEQ ID NO:40 or SEQ ID NO:41 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in SEQ ID NO:40 or SEQ ID NO:41. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:40, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO:40, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:40. In some embodiments, the target site is a contiguous portion of SEQ ID NO:40 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:40. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:41, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO:41, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:41. In some embodiments, the target site is a contiguous portion of SEQ ID NO:41 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:41.

[0260] In some embodiments, the gRNA targeting a target site for MYB comprises a spacer sequence set forth in SEQ ID NO:54 or SEQ ID NO:55, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:54 or SEQ ID NO:55 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ D NO:54 or SEQ ID NO:55. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:54, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:54. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:54. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:54. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:55, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:55. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:55. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:55.

[0261] In some embodiments, the gRNA targeting a target site for MYB 1 comprises a sequence comprising a spacer sequence set forth in SEQ ID NO:54 or SEQ ID NO:54 and further comprises a scaffold sequence, such as any scaffold sequence disclosed herein. In some embodiments, the gRNA targeting a target site for MYB1 comprises modified nucleotides.

[0262] In some embodiments, the gRNA targets a target site for RUNX1. In some embodiments, the gRNA targets the target site that comprises a sequence set forth in SEQ ID NO:42 or SEQ ID NO:43, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of SEQ ID NO:42 or SEQ ID NO:43 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in SEQ ID NO:42 or SEQ ID NO:43. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:42, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO:42, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:42. In some embodiments, the target site is a contiguous portion of SEQ ID NO:42 that is 14, 15, 16,17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:42. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:43, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO:43, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:43. In some embodiments, the target site is a contiguous portion of SEQ ID NO:43 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:43.

[0263] In some embodiments, the gRNA targeting a target site for RUNX1 comprises a spacer sequence set forth in SEQ ID NO:56 or SEQ ID NO:57, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:56 or SEQ ID NO:57 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ D NO:56 or SEQ ID NO:57. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:56, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:56. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:56. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:56. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:57, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:57. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:57. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:57.

[0264] In some embodiments, the gRNA targeting a target site for RUNX1 comprises a sequence comprising a spacer sequence set forth in SEQ ID NO:56 or SEQ ID NO:57and further comprises a scaffold sequence, such as any scaffold sequence disclosed herein. In some embodiments, the gRNA targeting a target site for RUNX1 comprises modified nucleotides.

[0265] In some embodiments, the gRNA targets a target site for HEY1. In someembodiments, the gRNA targets the target site that comprises a sequence set forth in SEQ ID NO:44 or SEQ ID NO:45, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of SEQ ID NO:44 or SEQ ID NO:45 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in SEQ ID NO:44 or SEQ ID NO:45. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:44, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO:44, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:44. In some embodiments, the target site is a contiguous portion of SEQ ID NO:44 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:44. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:45, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO:45, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:45. In some embodiments, the target site is a contiguous portion of SEQ ID NO:45 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:45.

[0266] In some embodiments, the gRNA targeting a target site for HEY 1 comprises a spacer sequence set forth in SEQ ID NO:58 or SEQ ID NO:59, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:58 or SEQ ID NO:59 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ D NO:58 or SEQ ID NO: 59. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:58, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:58. In some embodiments, the spacer sequence of the gRNA is a contiguousportion of SEQ ID NO:58. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:58. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:59, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:59. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:59. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:59.

[0267] In some embodiments, the gRNA targeting a target site for HEY 1 comprises a sequence comprising a spacer sequence set forth in SEQ ID NO:58 or SEQ ID NO:59 and further comprises a scaffold sequence, such as any scaffold sequence disclosed herein. In some embodiments, the gRNA targeting a target site for HEY1 comprises modified nucleotides.

[0268] In some embodiments, the gRNA targets a target site for RUNX3. In some embodiments, the gRNA targets the target site that comprises a sequence set forth in SEQ ID NO:46 or SEQ ID NO:47, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of SEQ ID NO:46 or SEQ ID NO:47 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in SEQ ID NO:46 or SEQ ID NO:47. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:46, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO:46, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:46. In some embodiments, the target site is a contiguous portion of SEQ ID NO:46 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:46. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:47, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO:47, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:47. In some embodiments, the target site is a contiguous portion of SEQ ID NO:47 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth inSEQ ID NO:47.

[0269] In some embodiments, the gRNA targeting a target site for RUNX3 comprises a spacer sequence set forth in SEQ ID NO: 60 or SEQ ID NO:61, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:60 or SEQ ID NO:61 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ D NO:60 or SEQ ID NO:61. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:60, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:60. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:60. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:60. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:61, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:61. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:61. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:61.

[0270] In some embodiments, the gRNA targeting a target site for RUNX3 comprises a sequence comprising a spacer sequence set forth in SEQ ID NO:60 or SEQ ID NO: 61 and further comprises a scaffold sequence, such as any scaffold sequence disclosed herein. In some embodiments, the gRNA targeting a target site for RUNX3 comprises modified nucleotides.

[0271] In some embodiments, the gRNA targets a target site for SPI1. In some embodiments, the gRNA targets the target site that comprises a sequence set forth in SEQ ID NO:48 or SEQ ID NO:49, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of SEQ ID NO:48 or SEQ ID NO:49 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in SEQ ID NO:48 or SEQ ID NO:49. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ IDNO:48, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO:48, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:48. In some embodiments, the target site is a contiguous portion of SEQ ID NO:48 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:48. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:49, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO:49, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:49. In some embodiments, the target site is a contiguous portion of SEQ ID NO:49 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:49.

[0272] In some embodiments, the gRNA targeting a target site for SPI1 comprises a spacer sequence set forth in SEQ ID NO:62 or SEQ ID NO:63, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:62 or SEQ ID NO:63 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ D NO: 62 or SEQ ID NO: 63. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:62, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:62. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:62. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:62. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:63, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:63. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:63. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:63.

[0273] In some embodiments, the gRNA targeting a target site for SPI1 comprises asequence comprising a spacer sequence set forth in SEQ ID NO:62 or SEQ ID NO:63 and further comprises a scaffold sequence, such as any scaffold sequence disclosed herein. In some embodiments, the gRNA targeting a target site for SPI1 comprises modified nucleotides.

[0274] In some embodiments, the gRNA targets a target site for ILR7a. In some embodiments, the gRNA targets the target site that comprises a sequence set forth in SEQ ID NO:50, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of SEQ ID NO:50 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in SEQ ID NO:50.

[0275] In some embodiments, the gRNA targeting a target site for ILR7a comprises a spacer sequence set forth in SEQ ID NO:64, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:64 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:64.

[0276] In some embodiments, the gRNA targeting a target site for ILR7a comprises a sequence comprising a spacer sequence set forth in SEQ ID NO:64 and further comprises a scaffold sequence, such as any scaffold sequence disclosed herein. In some embodiments, the gRNA targeting a target site for ILR7a comprises modified nucleotides.

[0277] In some embodiments, the gRNA targets a target site for TBX21. In some embodiments, the gRNA targets the target site that comprises a sequence set forth in SEQ ID NO:51, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of SEQ ID NO:51 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in SEQ ID NO:51.

[0278] In some embodiments, the gRNA targeting a target site for TBX21 comprises a spacer sequence set forth in SEQ ID NO: 65, or a contiguous portion thereof of at least 14 nt, or asequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:65 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:65.

[0279] In some embodiments, the gRNA targeting a target site for TBX21 comprises a sequence comprising a spacer sequence set forth in SEQ ID NO:65 and further comprises a scaffold sequence, such as any scaffold sequence disclosed herein. In some embodiments, the gRNA targeting a target site for TBX21 comprises modified nucleotides.

[0280] In some embodiments, the gRNA targets a target site for LEF1. In some embodiments, the gRNA targets the target site that comprises a sequence set forth in SEQ ID NO:52, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of SEQ ID NO:52 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in SEQ ID NO:52.

[0281] In some embodiments, the gRNA targeting a target site for LEF1 comprises a spacer sequence set forth in SEQ ID NO:66, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:66 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:66.

[0282] In some embodiments, the gRNA targeting a target site for LEF1 comprises a sequence comprising a spacer sequence set forth in SEQ ID NO:66 and further comprises a scaffold sequence, such as any scaffold sequence disclosed herein. In some embodiments, the gRNA targeting a target site for LEF1 comprises modified nucleotides.

[0283] In some embodiments, the gRNA targets a target site for CBFB. In some embodiments, the gRNA targets the target site that comprises a sequence set forth in SEQ ID NO:53, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of SEQ ID NO:53 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in SEQ ID NO:53.

[0284] In some embodiments, the gRNA targeting a target site for CBFB comprises a spacer sequence set forth in SEQ ID NO:67, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:67 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:67.

[0285] In some embodiments, the gRNA targeting a target site for CBFB comprises a sequence comprising a spacer sequence set forth in SEQ ID NO:67 and further comprises a scaffold sequence, such as any scaffold sequence disclosed herein. In some embodiments, the gRNA targeting a target site for CBFB comprises modified nucleotides.

[0286] In some embodiments, provided herein is a combination of gRNAs that each target a target site for a gene for transcriptional activation. In some embodiments, provided herein is a multiplexed DNA-targeting system comprising the combination of gRNAs.

[0287] In some embodiments, each gRNA of the combination of gRNAs is selected from any of the gRNAs described herein for targeted transcriptional activation. In some embodiments, the combination of gRNAs comprises a first gRNA targeted to a first gene and a second gRNA targeted to a second gene. In some embodiments, the combination of gRNAs comprises a gRNA targeted to DLL4. In some embodiments, the combination of gRNAs comprises a gRNA targeted to VCAM1. In some embodiments, the combination of gRNAs comprises a first gRNA targeted to DLL4 and a a second gRNA targeted to VCAM1. In some embodiments, the combination of gRNAs comprises at least three gRNAs targeting at least three different genes. In some embodiments, the combinations of gRNAs comprise a first gRNA targeted to DLL4, a second gRNA targeted to VCAM1, and one or more gRNAs targeted to genes selected from: MYB, RUNX3, RUNX1, SPI1, HEY1, IL7Ra, TBX21, LEF1, CBFB, TCF7, GATA3, and BCL1 IB. In some embodiments, the gRNAs are a combination of gRNAs that bind to target sites for any of the genes or any of the combinations of genes listed in Section I.B.D. Other DNA-Binding Domains and DNA-Targeting Systems

[0288] In some of any of the provided embodiments, the DNA-binding domain comprises a zinc finger protein (ZFP); a transcription activator-like effector (TALE); a meganuclease; a homing endonuclease; or an LScel enzyme or a variant thereof. In some embodiments, the DNA-binding domain comprises a catalytically inactive variant of any of the foregoing. In some embodiments, the fusion protein of the DNA-targeting system, or one or more DNA-targeting modules thereof, comprises a DNA-binding domain described herein, such as a DNA-binding domain that is an engineered zinc finger protein (eZFP) or a TALE.

[0289] In some embodiments, a ZFP, a zinc finger DNA binding protein, or zinc finger DNA binding domain, is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP. Among the ZFPs are artificial, or engineered ZFPs (eZFPs), comprising ZFP domains targeting specific DNA sequences, typically 9-18 nucleotides long, generated by assembly of individual fingers. ZFPs include those in which a single finger domain is approximately 30 amino acids in length and contains an alpha helix containing two invariant histidine residues coordinated through zinc with two cysteines of a single beta turn, and having two, three, four, five, or six fingers. Generally, sequence-specificity of a ZFP may be altered by making amino acid substitutions at the four helix positions (-1, 2, 3, and 6) on a zinc finger recognition helix. Thus, for example, the ZFP or ZFP-containing molecule is non-naturally occurring, e.g., is an eZFP that is engineered to bind to a target site of choice.

[0290] In some embodiments, zinc fingers are custom-designed (i.e. designed by the user), or obtained from a commercial source. Various methods for designing zinc finger proteins are available. For example, methods for designing zinc finger proteins to bind to a target DNA sequence of interest are described, for example in Liu, Q. et al., PNAS, 94(l l):5525-30 (1997); Wright, D.A. et al., Nat. Protoc., 1(3): 1637-52 (2006); Gersbach, C.A. et al., Acc. Chem. Res., 47(8):2309-18 (2014); Bhakta M.S. et al., Methods Mol. Biol., 649:3-30 (2010); and Gaj et al., Trends Biotechnol, 31(7):397-405 (2013). In addition, various web-based tools for designing zinc finger proteins to bind to a DNA target sequence of interest are publicly available. See, for example, the Zinc Finger Tools design web site from Scripps available on the world wide web at scripps.edu / barbas / zfdesign / zfdesignhome.php. Various commercial services for designing zincfinger proteins to bind to a DNA target sequence of interest are also available. See, for example, the commercially available services or kits offered by Creative Biolabs (world wide web at creative-biolabs.com / Design-and-Synthesis-of-Artificial-Zinc-Finger-Proteins.html), the Zinc Finger Consortium Modular Assembly Kit available from Addgene (world wide web at addgene.org / kits / zfc-modular-assembly / ), or the CompoZr Custom ZFN Service from Sigma Aldrich (world wide web at sigmaaldrich.com / life-science / zinc-finger-nuclease- technology / custom- zfn.html).

[0291] In some embodiments, the fusion protein of the DNA-targeting system comprises an eZFP DNA-binding domain and an effector domain.

[0292] Transcription activator-like effectors (TALEs), are proteins naturally found in Xanthomonas bacteria. TALEs comprise a plurality of repeated amino acid sequences, each repeat having binding specificity for one base in a target sequence. Each repeat comprises a pair of variable residues in position 12 and 13 (repeat variable diresidue; RVD) that determine the nucleotide specificity of the repeat. In some embodiments, RVDs associated with recognition of the different nucleotides are HD for recognizing C, NG for recognizing T, NI for recognizing A, NN for recognizing G or A, NS for recognizing A, C, G or T, HG for recognizing T, IG for recognizing T, NK for recognizing G, HA for recognizing C, ND for recognizing C, HI for recognizing C, HN for recognizing G, NA for recognizing G, SN for recognizing G or A and YG for recognizing T, TL for recognizing A, VT for recognizing A or G and SW for recognizing A. In some embodiments, RVDs can be mutated towards other amino acid residues in order to modulate their specificity towards nucleotides A, T, C and G and in particular to enhance this specificity. Binding domains with similar modular base-per-base nucleic acid binding properties can also be derived from different bacterial species. These alternative modular proteins may exhibit more sequence variability than TALE repeats.

[0293] In some embodiments, a “TALE DNA binding domain” or “TALE” is a polypeptide comprising one or more TALE repeat domains / units. The repeat domains, each comprising a repeat variable diresidue (RVD), are involved in binding of the TALE to its cognate target DNA sequence. A single “repeat unit” (also referred to as a “repeat”) is typically 33-35 amino acids in length and exhibits at least some sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. TALE proteins may be designed to bind to a target site using canonical or non-canonical RVDs within the repeat units. See, e.g., U.S. Pat. Nos. 8,586,526 and 9,458,205.

[0294] In some embodiments, the fusion protein of the DNA-targeting system comprises a TALE DNA-binding domain and an effector domain.

[0295] Zinc finger and TALE DNA-binding domains can be engineered to bind to a predetermined nucleotide sequence, for example via engineering (altering one or more amino acids) of the recognition helix region of a naturally occurring zinc finger protein, by engineering of the amino acids in a TALE repeat involved in DNA binding (the repeat variable diresidue or RVD region), or by systematic ordering of modular DNA-binding domains, such as TALE repeats or ZFP domains. Therefore, engineered zinc finger proteins or TALE proteins are proteins that are non-naturally occurring. Non-limiting examples of methods for engineering zinc finger proteins and TALEs are design and selection. A designed protein is a protein not occurring in nature whose design / composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP or TALE designs (canonical and non-canonical RVDs) and binding data. See, for example, U.S. Pat. Nos. 9,458,205; 8,586,526; 6,140,081; 6,453,242; and 6,534,261; see also WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536 and WO 03 / 016496.E. Effector Domains

[0296] In some aspects, the DNA-targeting systems provided herein further include one or more effector domains, such as a transcriptional activator effector domain. In some embodiments, provided herein is a DNA-targeting system comprising a fusion protein comprising: (a) a DNA-binding domain capable of being targeted to a target site in a gene or regulatory DNA element thereof, such as any DNA-binding domain described above in Section I.C or Section I.D, and (b) at least one effector domain. In some aspects, the effector domain is capable of increasing transcription of the gene, such as any of the genes described in Section I.B. In some aspects, the effector domain comprises a transcription activation domain.

[0297] In some aspects, the effector domain activates, induces, catalyzes, or leads to increased transcription of a gene when ectopically recruited to the gene or DNA regulatory element thereof. In some embodiments, the effector domain activates, induces, catalyzes, or leads to: transcription activation, transcription co-activation, transcription elongation, transcription de-repression, transcription factor release, polymerization, histone modification, histone acetylation, histone deacetylation, nucleosome remodeling, chromatin remodeling, reversal of heterochromatin formation, proteolysis, ubiquitination, deubiquitination,phosphorylation, dephosphorylation, DNA methylation, DNA demethylation, histone methylation, histone demethylation, or DNA base oxidation. In some embodiments, the effector domain activates, induces, catalyzes or leads to transcription activation, transcription coactivation, or transcription elongation. In some embodiments, the effector domain induces transcription activation. In some embodiments, the effector domain has one of the aforementioned activities itself (i.e. acts directly). In some embodiments, the effector domain recruits and / or interacts with a polypeptide domain that has one of the aforementioned activities (i.e. acts indirectly).

[0298] Gene expression of endogenous mammalian genes, such as human genes, can be achieved by targeting a fusion protein comprising a DNA-binding domain, such as a dCas9, and an effector domain, such as a transcription activation domain, to mammalian genes or regulatory DNA elements thereof (e.g. a promoter or enhancer) via one or more gRNAs. Any of a variety of effector domains for transcriptional activation (e.g. transcription activation domains) are known and can be used in accord with the provided embodiments. Transcription activation domains, as well as activation of target genes by Cas fusion proteins (with a variety of Cas molecules) and the transcription activation domains, are described, for example, in WO 2014 / 197748, WO 2016 / 130600 , WO 2017 / 180915, WO 2021 / 226555 , WO 2021 / 226077, WO 2013 / 176772 , WO 2014 / 152432, WO 2014 / 093661, WO 2024 / 015881, Adli, M. Nat. Commun. 9, 1911 (2018), Perez-Pinera, P. et al. Nat. Methods 10, 973-976 (2013), Mali, P. et al. Nat. Biotechnol. 31, 833-838 (2013), and Maeder, M. L. et al. Nat. Methods 10, 977-979 (2013), the disclosures of which are incorporated by reference in their entirety.

[0299] In some embodiments, a transcriptional activation domain comprises a domain of a protein selected from among VP64, p65, Rta, p300, CBP, VPR, VPH, HSF1, a TET protein (e.g. TET1), a partially or fully functional fragment or domain thereof, or a combination of any of the foregoing. In some embodiments, a transcriptional activator domain further comprises at least one domain of a protein selected from among FOXO3 and NCOA3, that exhibits transcriptional activation, is capable of inducing or activating transcription from a gene, is a functional transcriptional activation domain, and / or has a function of transcription activation. In some embodiments, a transcriptional activator domain further comprises at least one domain selected from among FOXO3 and NCOA3.

[0300] In some embodiments, the transcriptional activation domain comprises a VP64 domain. For example, dCas9-VP64 can be targeted to a target site by one or more gRNAs toactivate a gene. VP64 is a polypeptide composed of four tandem copies of VP 16, a 16 amino acid transactivation domain of the Herpes simplex virus. VP64 domains, including in dCas fusion proteins, have been described, for example, in WO 2014 / 197748, WO 2013 / 176772, WO 2014 / 152432, and WO 2014 / 093661. In some embodiments, the transcriptional activation domain comprises at least one VP 16 domain, or a VP 16 tetramer (“VP64”) or a variant thereof. In some embodiments, the transcriptional activation domain comprises SEQ ID NO:28, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:28, or a portion thereof. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO:28. In some embodiments, the transcriptional activation domain comprises SEQ ID NO:30, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:30, or a portion thereof. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO:30.

[0301] In some embodiments, the transcriptional activation domain comprises a p65 activation domain (p65AD). p65AD is the principal transactivation domain of the 65kDa polypeptide of the nuclear form of the NF-KB transcription factor. An exemplary sequence of human transcription factor p65 is available at the Uniprot database under accession number Q04206. P65 domains, including in dCas fusion proteins, have been described, for example in WO 2017 / 180915 and Chavez, A. et al. Nat. Methods 12, 326-328 (2015). An exemplary p65 activation domain is set forth in SEQ ID NO: 187. In some embodiments, the transcriptional activation domain comprises SEQ ID NO: 187, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 187, or a portion thereof. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO: 187.

[0302] In some embodiments, the transcriptional activation domain comprises an R transactivator (Rta) domain. Rta is an immediate-early protein of Epstein-Barr virus (EBV), and is a transcriptional activator that induces lytic gene expression and triggers virus reactivation. The Rta domain, including in dCas fusion proteins, has been described, for example in WO 2017 / 180915 and Chavez, A. et al. Nat. Methods 12, 326-328 (2015). An exemplary Rta domain is set forth in SEQ ID NO: 187. In some embodiments, the transcriptional activation domain comprises SEQ ID NO: 187, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 187, or a portion thereof. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO:187.

[0303] In some embodiments, the transcriptional activation domain comprises a CREB- binding protein (CBP) domain or a p300 domain. In some aspects, CBP refers to the CREB- binding protein encoded by the human CREBBP gene. CBP is a coactivator that interacts with cAMP-response element binding protein (CREB). In some aspects, p300 refers to the Histone acetyltransferase p300 protein encoded by the human EP300 gene, and is a coactivator closely related to CBP. CBP and p300 each interact with a variety of transcriptional activators to affect gene transcription (Gerritsen, M.E. et al. PNAS 94(7):2927-2932 (1997)). In some embodiments, the transcriptional activation domain comprises a p300 domain. P300 domains (such as the catalytic core of p300) including in dCas fusion proteins for gene activation, has been described, for example, in WO 2016 / 130600, WO 2017 / 180915, and Hilton, I.B. et al., Nat. Biotechnol. 33(5):510-517 (2015). . An exemplary human CBP sequence is set forth in SEQ ID NO: 189. An exemplary human p300 sequence is set forth in SEQ ID NO: 190. An exemplary p300 domain is set forth in SEQ ID NO: 191. In some embodiments, the transcriptional activation domain comprises any one of SEQ ID NOS: 189- 191, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequ...

Claims

1. CLAIMSWHAT IS CLAIMED:

1. A DNA-targeting system comprising a DNA-targeting module, wherein the DNA-targeting module comprises:(a) a first DNA-targeting module for increasing transcription of a DLL4 gene, wherein the DNA-targeting module comprises a fusion protein comprising (i) a DNA- binding domain that binds to a target site for DLL4, and (ii) at least one transcriptional activator effector domain; or(b) a second DNA-targeting module for increasing transcription of a VCAM1 gene, wherein the DNA-targeting module comprises a fusion protein comprising (i) a DNA-binding domain that binds to a target site for VCAM1, and (ii) at least one transcriptional activator effector domain.

2. A DNA-targeting system comprising a plurality of DNA-targeting modules, wherein the plurality of DNA-targeting modules comprises:(a) a first DNA-targeting module for increasing transcription of a DLL4 gene, wherein the first DNA-targeting module comprises a fusion protein comprising (i) a DNA-binding domain that binds to a target site for DLL4, and (ii) at least one transcriptional activator effector domain; and(b) a second DNA-targeting module for increasing transcription of a VCAM1 gene, wherein the second DNA-targeting module comprises a fusion protein comprising (i) a DNA-binding domain that binds to a target site for VCAM1, and (ii) at least one transcriptional activator effector domain.

3. The DNA-targeting system of claim 1 or claim 2, wherein the DNA-targeting system does not introduce a genetic disruption or a DNA break.

4. The DNA-targeting system of any of claims 1-3, wherein the fusion protein of the first DNA-targeting module and / or the second DNA-targeting module comprises a DNA- binding domain selected from: a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or a variant thereof; a zinc finger protein (ZFP); a transcriptionactivator-like effector (TALE); a meganuclease; a homing endonuclease; or an LScel enzyme or a variant thereof, optionally wherein the DNA-binding domain comprises a catalytically inactive variant of any of the foregoing, wherein, when the DNA-binding domain of the fusion protein comprises a Cas protein, the first DNA-targeting module comprises a first guide nucleic acid for targeting the Cas protein to the target site of the DLL4 gene and / or the second DNA-targeting module comprises a second guide nucleic acid for targeting the Cas protein to the target site of the VCAM1 gene.

5. The DNA-targeting system of any one of claims 1-4, wherein the DNA-binding domain is a zinc finger protein.

6. The DNA-targeting system of any one of claims 1-5, comprising at least the first and second DNA-targeting module, wherein the fusion protein of each of the plurality of DNA- targeting modules is different.

7. The DNA-targeting system of any of claims 1-4, comprising at least the first and second DNA-targeting module, wherein the DNA-targeting system comprises one fusion protein that is shared by each of the DNA-targeting modules and wherein each DNA-targeting module is characterized by comprising a different guide nucleic acid for targeting the DNA-binding domain to its respective target site.

8. The DNA-targeting system of claim 7, wherein the first DNA-targeting molecule comprises a first guide nucleic acid for targeting a target site for DLL4 and the second DNA- targeting molecule comprises a second guide nucleic acid for targeting a target site for VCAM1.

9. The DNA-targeting system of claim 7 or 8, wherein the DNA-binding domain of the fusion protein is a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof.

10. The DNA-targeting system of any of claims 1-4, 8 and 9, wherein the DNA- binding domain of the first DNA-targeting module and the second DNA-targeting module is a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variantthereof, and the first DNA-targeting module comprises at least a first guide nucleic acid for targeting the Cas protein to the target site for DLL4 and the second DNA-targeting module comprises at least a second guide nucleic acid for targeting the Cas protein to the target site for VCAM1.

11. The DNA-targeting system of any of claims 4 and 7-10, wherein the guide nucleic acid is a guide RNA (gRNA), optionally wherein the first guide nucleic acid is a first gRNA and the second guide nucleic acid is a second gRNA.

12. The DNA-targeting system of any of claims 4 and 7-11, wherein the Cas protein or variant thereof is a deactivated (dCas) protein.

13. A DNA targeting system comprising:(a) a fusion protein comprising a DNA-binding domain comprising a deactivated Cas (dCas) protein and at least one transcriptional activator effector domain;(b) a plurality of gRNAs comprising a first gRNA that targets a target site for DLL4 and a second gRNA that targets a target site for VCAM1.

14. The DNA-targeting system of claim 13, wherein the DNA-targeting system increases transcription of the DLL4 gene and increases transcription of the VCAM1 gene.

15. The DNA-targeting system of claim 12 or claim 13, wherein the dCas protein lacks nuclease activity.

16. The DNA-targeting system of any one of claims 1-15, wherein the target site for DLL4 is in the DLL4 gene or a regulatory DNA element thereof.

17. The DNA-targeting system of claim 16, wherein the regulatory DNA element is an enhancer or a promoter of the gene.

18. The DNA-targeting system of claim 17, wherein the regulatory DNA element is a promoter of the gene.

19. The DNA-targeting system of any of claims 1-18, wherein the target site forDLL4 is within the genomic coordinates chrl5:40,905,000 to chrl5:40,943,500.

20. The DNA-targeting system of any of claims 1-19, wherein the target site for DLL4 is within the genomic coordinates chrl5: 40,905,000 to chrl 5:40,909,000; chrl5: 40,925,250 to chrl5:40,934,00; chrl5: 40,937,800 to chrl5:40,938,800; or chrl5: 40,940,000 to chrl5:40,943,500.

21. The DNA-targeting system of any of claims 1-20, wherein the target site for DLL4 is within the genomic coordinates chrl5: 40,925,250 to chrl5:40,934,00.

22. The DNA-targeting system of any of claims 1-20, wherein the target site for DLL4 is within the genomic coordinates chrl5: 40,928,340 to chrl5:40,930,340.

23. The DNA-targeting system of any of claims 1-18, wherein the target site for DLL4 is within 1000 base pairs of the transcription start site (TSS) of the gene.

24. The DNA-targeting system of any of claims 1-23, wherein the target site for DLL4 is within 20 base pairs, 50 base pairs, 100 base pairs, 200 base pairs, 300 base pairs, 400 base pairs, 500 base pairs, 600 base pairs, or any value between any of the foregoing, of the TSS of the gene.

25. The DNA-targeting system of any of claims 1-24, wherein the target site for DLL4 is within 550 base pairs upstream of the TSS of the gene.

26. The DNA-targeting system of any of claims 1-25, wherein the target site for DLL4 is within 250 base pairs upstream of the TSS of the gene.

27. The DNA-targeting system of any of claims 1-25, wherein the target site for DLL4 is within the genomic coordinates chrl5: 40,929,100 to chrl5:40,929,170.

28. The DNA-targeting system of any of claims 1-27, wherein the target site forDLL4 is a target site having the sequence set forth in any one of SEQ ID NOs:l, 2 and 147-166, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

29. The DNA-targeting system of any of claims 1-27, wherein the target site for DLL4 is a target site having the sequence set forth in SEQ ID NO:1 or SEQ ID NO: 2, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

30. The DNA-targeting system of any of claims 1-29, wherein the target site for DLL4 is a target site having the sequence set forth in SEQ ID NO:1 or SEQ ID NO: 2.

31. The DNA-targeting system of any of claims 1-29, wherein the target site for DLL4 is a target site having the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 154.

32. The DNA-targeting system of any of claims 1-31, wherein the target site for DLL4 has the sequence set forth in SEQ ID NO: 154 or a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence thereof.

33. The DNA-targeting system of any of claims 1-32, wherein the target site for DLL4 has the sequence set forth in SEQ ID NO: 154.

34. The DNA-targeting system of any of claims 1-31, wherein the target site for DLL4 has the sequence set forth in SEQ ID NO:2 or a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence thereof.

35. The DNA-targeting system of any of claims 1-31 or 34, wherein the target site for DLL4 has the sequence set forth in SEQ ID NO:2.

36. The DNA-targeting system of any of claims 1-35, wherein the target site for VCAM1 is in the VC AMI gene or a regulatory DNA element thereof.

37. The DNA-targeting system of claim 36, wherein the regulatory DNA element is an enhancer or a promoter of the gene.

38. The DNA-targeting system of claim 37, wherein the regulatory DNA element is a promoter of the gene.

39. The DNA-targeting system of any of claims 1-38, wherein the target site for VCAM1 is within 1000 base pairs of the transcription start site (TSS) of the gene.

40. The DNA-targeting system of any of claims 1-39, wherein the target site for VCAM1 is within 20 base pairs, 50 base pairs, 100 base pairs, 200 base pairs, 300 base pairs, 400 base pairs, 500 base pairs, 600 base pairs, or any value between any of the foregoing, of the TSS of the gene.

41. The DNA-targeting system of any of claims 1-40, wherein the target site for VCAM1 is within 550 base pairs upstream of the TSS of the gene.

42. The DNA-targeting system of any of claims 1-41, wherein the target site for VCAM1 is within the genomic coordinates chrl:100,719,182 to chrl: 100,720,290.

43. The DNA-targeting system of any of claims 1-42, wherein the target site for VCAM1 is a target site having the sequence set forth in SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

44. The DNA-targeting system of any of claims 1-43, wherein the target site for VCAM1 is a target site having the sequence set forth in SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.

45. The DNA-targeting system of any of claims 1-44, wherein the target site forVCAM1 has the sequence set forth in SEQ ID NO: 3 or a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence thereof.

46. The DNA-targeting system of any of claims 1-45, wherein the target site for VCAM1 has the sequence set forth in SEQ ID NO: 3.

47. The DNA-targeting system of any of claims 11-46, wherein the first gRNA comprises a gRNA spacer that is complementary to the target site of DLL4.

48. The DNA-targeting system of any of claims 11-47, wherein the first gRNA targeting a target site for DLL4 comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 8, 9, and 167-186, or a contiguous portion thereof of at least 14 nt.

49. The DNA-targeting system of any of claims 11-48, wherein the first gRNA targeting a target site for DLL4 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 174, or a contiguous portion thereof of at least 14 nt.

50. The DNA-targeting system of any of claims 11-49, wherein the first gRNA targeting a target site for DLL4 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 174.

51. The DNA-targeting system of any of claims 11-50, wherein the first gRNA targeting a target site for DLL4 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9, or a contiguous portion thereof of at least 14 nt.

52. The DNA-targeting system of any of claims 11-51, wherein the first gRNA targeting a target site for DLL4 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9.

53. The DNA-targeting system of any of claims 11-52, wherein the first gRNA targeting a target site for DLL4 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 9.

54. The DNA-targeting system of any of claims 11-53, wherein the second gRNA comprises a gRNA spacer that is complementary to the target site of VCAM1.

55. The DNA-targeting system of any of claims 1-54, wherein the second gRNA targeting a target site of VCAM1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, or a contiguous portion thereof of at least 14 nt.

56. The DNA-targeting system of any of claims 11-54, wherein the second gRNA targeting a target site of VCAM1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14.

57. The DNA-targeting system of any of claims 11-56, wherein the second gRNA targeting a target site for VCAM1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 10.

58. The DNA targeting system of any of claims 1-57, wherein the DNA-targeting modules further comprise one or more additional DNA-targeting modules for increasing transcription of one or more lymphoid cell differentiation (LCD) genes, wherein each of the one or more additional DNA-targeting modules comprises a fusion protein comprising:(a) a DNA-binding domain that binds to a target site for one of the one or more LCD genes; and(b) at least one transcriptional activator effector domain.

59. The DNA-targeting system of claim 58, wherein the DNA-binding domain of each of the one or more additional DNA-targeting modules is a deactivated Cas (dCas) protein,and wherein each of the one or more additional DNA-targeting module comprises a guide nucleic acid for targeting the Cas protein to the target site of one of the one or more LCD genes.

60. The DNA-targeting system of claim 58 or claim 59, wherein the DNA-targeting system comprises a plurality of DNA-targeting modules comprising the first and / or second DNA-targeting module and the one or more additional DNA-targeting modules, wherein the DNA-targeting system comprises one fusion protein that is shared by each of the plurality of DNA-targeting modules and wherein each DNA-targeting module is characterized by comprising a different guide nucleic acid for targeting the DNA-binding domain to its respective target site, optionally wherein each different guide nucleic acid is a gRNA.

61. The DNA-targeting system of any of claims 58-60, wherein the DNA-targeting system comprises:(1) a fusion protein comprising (a) a deactivated Cas (dCas) protein; and (b) at least one transcriptional activator effector domain;(2) a guide nucleic acid, optionally a gRNA, for targeting the dCas protein to the target site for DLL4;(3) a guide nucleic acid, optionally a gRNA, for targeting the dCas protein to the target site for VCAM1; and(4) one or more additional guide nucleic acids, optionally at least one or more additional gRNA, for targeting the Cas protein to the target site of each of the one or more LCD genes.

62. The DNA-targeting system of claim 61, wherein the guide nucleic acid is a gRNA.

63. The DNA-targeting system of claim 61, wherein the DNA-targeting system comprises:(1) a fusion protein comprising (a) a deactivated Cas (dCas) protein; and (b) at least one transcriptional activator effector domain;(2) a gRNA targeting a target site for DLL4;(3) a gRNA targeting a target site for VCAM1; and(4) one or more gRNA targeting a target site of the one or more LCD genes, optionally wherein each of the one or more gRNA targets a target site of one of the LCD genes.

64. The DNA-targeting system of claim 61 or claim 63, wherein the DNA-targeting system comprises 3 to 10 different gRNAs, optionally 3 to 6 different gRNAs.

65. The DNA-targeting system of any of claims 58-64, wherein the one or more LCD genes is selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL11B.

66. The DNA-targeting system of claim 58 or claim 65, wherein the one or more additional DNA-targeting modules is one additional DNA-targeting module, wherein the one additional DNA-targeting module targets a target site of a LCD gene selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL11B.

67. The DNA-targeting system of claim 58 or claim 65, wherein the one or more additional DNA-targeting modules target two or more LCD genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL11B.

68. The DNA-targeting system of any of claims 58-67, wherein the LCD genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

69. The DNA-targeting system of any of claims 65-68, wherein at least one LCD gene is RUNX3.

70. The DNA-targeting system of any of claims 61-69, wherein the DNA-targeting system comprises:(1) a fusion protein comprising (a) a deactivated Cas (dCas) protein; and (b) at least one transcriptional activator effector domain;(2) a gRNA targeting a target site for DLL4;(3) a gRNA targeting a target site for VCAM1; and(4) a gRNA targeting a target site for RUNX3.

71. The DNA-targeting system of any of claims 65-68, wherein at least one LCD gene is RUNX1.

72. The DNA-targeting system of any of claims 65-68, wherein at least one LCD gene is LEF1.

73. The DNA-targeting system of any of claims 65-68, wherein at least one LCD gene is MYB.

74. The DNA-targeting system of claim 65, wherein at least one LCD gene is TCF7.

75. The DNA-targeting system of claim 65, wherein at least one LCD gene is BCL11B.

76. The DNA-targeting system of claim 65, wherein the one or more LCD genes are TCF7, BCL11B, and MYB.

77. The DNA-targeting system of claim 65, wherein the LCD genes are TCF7, MYB, and RUNX3.

78. The DNA-targeting system of any of claims 58-77, wherein the target site for each of the one or more LCD genes is in the gene or a regulatory DNA element thereof.

79. The DNA-targeting system of claim 78, wherein the regulatory DNA element is an enhancer or a promoter of the gene.

80. The DNA-targeting system of claim 79, wherein the regulatory DNA element is a promoter of the gene.

81. The DNA-targeting system of any of claims 58-80, wherein the target site for each of the one or more LCD genes is within 1000 base pairs of the transcription start site (TSS) of the gene.

82. The DNA-targeting system of any of claims 58-81, wherein the target for each of the one or more LCD genes is within 20 base pairs, 50 base pairs, 100 base pairs, 200 base pairs, 300 base pairs, 500 base pairs, 600 base pairs, or any value between any of the foregoing, of the TSS of the gene.

83. The DNA-targeting system of any of claims 58-82, wherein the target site for each of the one or more LCD genes is within 550 base pairs of the TSS of the gene.

84. The DNA-targeting system of any of claims 58-70 and 77-83, wherein the target site for RUNX3 has the sequence forth in SEQ ID NO: 46 or SEQ ID NO: 47, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

85. The DNA-targeting system of any of claims 58-70 and 77-83, wherein the target site for RUNX3 has the sequence forth in SEQ ID NO: 46.

86. The DNA-targeting system of any of claims 58-70 and 77-83, wherein the gRNA targeting a target site for RUNX3 comprises a gRNA spacer sequence comprising the sequence forth in SEQ ID NO: 60 or SEQ ID NO: 61, or a contiguous portion thereof of at least 14 nucleotides (nt).

87. The DNA-targeting system of any of claims 58-70 and 77-83, wherein the gRNA spacer sequence comprises the sequence forth in SEQ ID NO: 60.

88. The DNA-targeting system of any of claims 12-87, wherein the dCas protein is a dCas9 protein.

89. The DNA-targeting system of any of claims 12-88, wherein the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein.

90. The DNA-targeting system of claim 89, wherein the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 88.

91. The DNA-targeting system of claim 89 or claim 90, wherein the dSaCas9 protein comprises the sequence set forth in SEQ ID NO: 89, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

92. The DNA-targeting system of any of claims 89-91, wherein the dSaCas9 protein is set forth in SEQ ID NO: 89.

93. The DNA-targeting system of any of claims 12-87, wherein the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein.

94. The DNA-targeting system of claim 93, wherein the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO: 31.

95. The DNA-targeting system of claim 93 or claim 94, wherein the dSpCas9 protein comprises the sequence set forth in SEQ ID NO: 32, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

96. The DNA-targeting system of any of claims 93-95, wherein the dSpCas9 protein is set forth in SEQ ID NO: 32.

97. The DNA-targeting system of any of claims 11-96, wherein each gRNA independently comprises a spacer sequence between 14 nt and 24 nt, or between 16 nt and 22 nt in length.

98. The DNA-targeting system of any of claims 11-97, wherein each gRNA independently comprises a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

99. The DNA-targeting system of any of claims 11-88 and 93-98, wherein each gRNA independently further comprises a scaffold sequence set forth in SEQ ID NO: 83.

100. The DNA-targeting system of any of claims 11-88 and 93-98, wherein each gRNA independently further comprises a scaffold sequence set forth in SEQ ID NO: 23.

101. The DNA-targeting system of any of claims 11-100, wherein each gRNA further comprises 2’ MeO modified bases and / or phosphorothiate backbone modifications.

102. The DNA-targeting system of any of claims 1-101, wherein the at least one transcriptional activator effector domain is selected from the group consisting of: a VP64 domain, a p65 activation domain, a p300 domain, an Rta domain, a CBP domain, a VPR domain, a VPH domain, an HSF1 domain, a TET protein domain, optionally wherein the TET protein is TET1, a SunTag domain, or a domain, portion, variant, or truncation of any of the foregoing.

103. The DNA-targeting system of any of claims 1-102, wherein the at least one transcriptional activator effector domain comprises at least one VP 16 domain, and / or a VP 16 tetramer (“VP64”) or a variant thereof.

104. The DNA-targeting system of any of claims 1-103, wherein the at least one transcriptional activator effector domain comprises a VP64 domain or a variant or portion thereof that exhibits transcriptional activation activity.

105. The DNA-targeting system of any of claims 1-104, wherein the at least one transcriptional activator effector domain is VP64.

106. The DNA-targeting system of claim 105, wherein the VP64 is positioned N- terminal and / or C-terminal to the DNA-binding domain. 16107. The DNA-targeting system of any of claims 1-106, wherein the at least one transcriptional activator effector domain comprises two copies of VP64.

108. The DNA-targeting system of any of claims 1-107, wherein the at least one transcriptional activator effector domain comprises the amino acid sequence set forth in SEQ ID NO: 28, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 28.

109. The DNA-targeting system of any of claims 1-108, wherein the at least one transcriptional activator effector domain comprises the amino acid sequence set forth in SEQ ID NO: 30, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 30.

110. The DNA-targeting system of any of claims 1-88 and 93-109, wherein the fusion protein comprises the sequence set forth in SEQ ID NO: 26, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

111. The DNA-targeting system of any of claims 1-110, wherein increasing transcription of DLL4 and VCAM1, and optionally increasing transcription of the one or more LCD genes, promotes differentiation of a hematopoietic progenitor cell (HPC) to a lymphoid progenitor.

112. The DNA-targeting system of any of claims 1-111, wherein transient delivery of the DNA-targeting system to a hematopoietic progenitor cell (HPC) promotes differentiation to a lymphoid progenitor.

113. The DNA targeting system of claim 112, wherein the lymphoid progenitor is an induced common lymphoid progenitor (iCLP).

114. The DNA-targeting system of any of claims 1-113, wherein transient delivery of the DNA-targeting system to an HPC promotes differentiation to a CD5+ cell.

115. The DNA-targeting system of any of claims 1-114, wherein transient delivery of the DNA-targeting system to an HPC promotes differentiation to a CD7+ cell.

116. The DNA-targeting system of any of claims 1-115, wherein transient delivery of the DNA-targeting system to an HPC promotes differentiation to a CD5+ / CD7+ cell.

117. The DNA-targeting system of any of claims 111-116, wherein the HPC is an induced hematopoietic progenitor cell (iHPC).

118. A gRNA that targets a target site for DLL4, wherein the target site for DLL4 comprises the sequence set forth in any one of SEQ ID NOs:l-2 and 147-166, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

119. The gRNA of claim 118, wherein the target site for DLL4 comprises the sequence set forth SEQ ID NO: 154, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

120. The gRNA of claim 118 or 119, wherein the target site for DLL4 comprises the sequence set forth SEQ ID NO: 154.

121. The gRNA of claim 118, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 8,9 and 167-186, or a contiguous portion thereof of at least 14 nt.

122. The gRNA of claim 121, wherein the gRNA spacer sequence comprises the sequence set forth in SEQ ID NO: 174, or a contiguous portion thereof of at least 14 nt.

123. The gRNA of claim 118, wherein the target site for DLL4 comprises the sequence set forth in any one of SEQ ID NOS: 1-2 a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

124. The gRNA of claim 123, wherein the target site for DLL4 comprises the sequence set forth in any one of SEQ ID NOS: 1-2.

125. The gRNA of claim 123 or claim 124, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9, or a contiguous portion thereof of at least 14 nt.

126. The gRNA of claim 125, wherein the gRNA spacer sequence comprises the sequence set forth in SEQ ID NO: 8 or SEQ ID NO:9.

127. A gRNA that targets a target site for VCAM1, wherein the target site for VC AM comprises the sequence set forth in any one of SEQ ID NOS:3-7 a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

128. The gRNA of claim 127, wherein the target site for VCAM comprises the sequence set forth in any one of SEQ ID NOS:3-7.

129. The gRNA of claim 127 or claim 128, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, or a contiguous portion thereof of at least 14 nt.

130. The gRNA of claim 129, wherein the gRNA spacer sequence comprises the sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14.

131. The gRNA of any of claims 123-130, wherein the spacer sequence is between 14 nt and 24 nt, or between 16 nt and 22 nt in length.

132. The gRNA of any of claims 123-131, wherein the spacer sequence is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

133. The gRNA of any of claims 123-132, wherein the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 23.

134. The gRNA of any of claims 123-132, wherein the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 83.

135. The gRNA of any of claims 123-133, wherein the gRNA further comprises 2’ MeO modified bases and / or phosphorothiate backbone modifications.

136. A combination comprising two or more gRNAs of any of claims 123-135.

137. The combination of claim 136, wherein at least one gRNA targets a target site in DLL4 and at least one gRNA targets a target site in VCAM1.

138. A combination of gRNAs comprising a first gRNA that targets a target site for DLL4 and a second gRNA that targets a target site for VCAM1.

139. The combination of gRNAs of claim 138, wherein the target site for DLL4 is within DLL4 or a regulatory element thereof.

140. The combination of gRNAs of claim 138 or claim 139, wherein the target site for VC AMI is within VCAM1 or a regulatory element thereof.

141. The combination of gRNAs of claim 139 or 140, wherein the regulatory DNA element is an enhancer or a promoter.

142. The combination of gRNAs of any of claims 139, 140, and 141, wherein the regulatory DNA element is a promoter.

143. The combination of gRNAs of any of claims 139-142, wherein the target site for DLL4 is within an enhancer or promoter.

144. The combination of gRNAs of any of claims 139-143, wherein the target site for DLL4 is within 1000 base pairs of the transcription start site (TSS) of DLL4.

145. The combination of gRNAs of any of claims 139-144, wherein the target site for DLL4 is within 20 base pairs, 50 base pairs, 100 base pairs, 200 base pairs, 300 base pairs, 400 base pairs, 500 base pairs, 600 base pairs, or any value between any of the foregoing, of the TSS.

146. The combination of gRNAs of any of claims 139-145, wherein the target site for DLL4 and the target site for VCAM1 is with 550 base pairs upstream of each TSS.

147. The combination of gRNAs of any of claims 139-146, wherein the target site for DLL4 has a sequence set forth in any one of SEQ ID NOs:l, 2 or 147-166, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

148. The combination of gRNAs of any of claims 139-147, wherein the target site for DLL4 has a sequence set forth in SEQ ID NO: 154 or a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence thereof.

149. The combination of gRNAs of any of claims 139-148, wherein the target site for DLL4 has a sequence set forth in SEQ ID NO: 154.

150. The combination of gRNAs of any of claims 139-149, wherein the target site for DLL4 has a sequence set forth in SEQ ID NO:1 or SEQ ID NO: 2, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

151. The combination of gRNAs of any of claims 139-150, wherein the target site for DLL4 has a sequence set forth in SEQ ID NO:1 or SEQ ID NO: 2.

152. The combination of gRNAs of any of claims 139-151, wherein the target site for DLL4 has a sequence set forth in SEQ ID NO:2 or a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence thereof.

153. The combination of gRNAs of any of claims 139-152, wherein the target site for DLL4 has a sequence set forth in SEQ ID NO:2.

154. The combination of gRNAs of any of claims 139-153, wherein the first gRNA comprises a gRNA spacer that is complementary to the target site of DLL4.

155. The combination of gRNAs of any of claims 139-154, wherein the first gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 174, or a contiguous portion thereof of at least 14 nt.

156. The combination of gRNAs of any of claims 139-155, wherein the first gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 174.

157. The combination of gRNAs of any of claims 139-154, wherein the first gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9, or a contiguous portion thereof of at least 14 nt.

158. The combination of gRNAs of any of claims 139-156, wherein the first gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9.

159. The combination of gRNAs of any of claims 139-157, wherein the first gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 9.

160. The combination of gRNAs of any of claims 139-159, wherein the target site for VC AMI is within an enhancer or promoter.

161. The combination of gRNAs of any of claims 139-160, wherein the target site for VCAM1 is within 1000 base pairs of the transcription start site (TSS) of VCAM1.

162. The combination of gRNAs of any of claims 139-161, wherein the target site for VCAM1 is within 20 base pairs, 50 base pairs, 100 base pairs, 200 base pairs, 300 base pairs, 400 base pairs, 500 base pairs, 600 base pairs, or any value between any of the foregoing, of the TSS.

163. The combination of gRNAs of any of claims 139-162, wherein the target site for VCAM1 is with 550 base pairs upstream of the TSS.

164. The combination of gRNAs of any of claims 139-163, wherein the target site for VCAM1 has a sequence set forth in SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

165. The combination of gRNAs of any of claims 139-164, wherein the target site for VCAM1 has a sequence set forth in SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

166. The combination of gRNAs of any of claims 139-165, wherein the target site for VCAM1 has a sequence set forth in SEQ ID NO:3 or a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence thereof.

167. The combination of gRNAs of any of claims 139-166, wherein the target site for VCAM1 has a sequence set forth in SEQ ID NO:3.

168. The combination of gRNAs of any of claims 139-167, wherein the second gRNA comprises a gRNA spacer that is complementary to the target site of VCAM1.

169. The combination of gRNAs of any of claims 139-168, wherein the second gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or a contiguous portion thereof of at least 14 nt.

170. The combination of gRNAs of any of claims 139-169, wherein the second gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14.

171. The combination of gRNAs of any of claims 139-170, wherein the second gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 10.

172. The combination of gRNAs of any of claims 139-171, wherein the first gRNA and the second gRNA independently comprise a spacer sequence between 14 nt and 24 nt, or between 16 nt and 22 nt in length.

173. The combination of gRNAs of any of claims 139-172, wherein the first gRNA and the second gRNA independently comprise a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

174. The combination of gRNAs of any of claims 139-173, wherein the first gRNA and the second gRNA each further comprise a scaffold sequence set forth in SEQ ID NO: 23.

175. The combination of gRNAs of any of claims 139-173, wherein the first gRNA and the second gRNA each further comprise a scaffold sequence set forth in SEQ ID NO: 83.

176. The combination of gRNAs of any of claims 139-175, wherein the first gRNA and / or the second gRNA independently further comprise 2’ MeO modified bases and / or phosphorothiate backbone modifications.

177. The combination of gRNAs of any of claims 139-176, further comprising one or more additional gRNAs, wherein the one or more additional gRNAs target a target site of one or more lymphoid cell differentiation (LCD) genes, wherein the one or more LCD genes is selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL11B.

178. The combination of gRNAs of claim 177, wherein the one or more additional gRNAs is one additional gRNA, wherein the one additional gRNA targets a target site of an LCD gene selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL11B.

179. The combination of gRNAs of claim 178, wherein the additional gRNA targets a target site of RUNX3.

180. The combination of gRNAs of claim 179, wherein the target site for RUNX3 has the sequence forth in SEQ ID NO: 46 or SEQ ID NO: 47, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

181. The combination of gRNAs of claim 179 or claim 180, wherein the target site for RUNX3 has the sequence forth in SEQ ID NO: 46.

182. The combination of gRNAs of claim 177, wherein the one or more additional gRNAs are two gRNAs, wherein the two additional gRNAs target a target sites of two or more LCD genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL11B, or any combination thereof.

183. The combination of gRNAs of any of claims 177-182, wherein the LCD genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

184. The combination of gRNAs of any of claims 177-183, wherein at least one LCD gene is RUNX3.

185. The combination of gRNAs of any of claims 177-183, wherein at least one LCD gene is RUNX1.

186. The combination of gRNAs of any of claims 177-183, wherein at least one LCD gene is LEF1.

187. The combination of gRNAs of claim 182 or claim 183, wherein at least one LCD gene is MYB.

188. The combination of gRNAs of claim 182, wherein at least one LCD gene is TCF7.

189. The combination of gRNAs of claim 182, wherein at least one LCD gene is BCL11B.

190. The combination of gRNAs of claim 182, wherein the one or more LCD genes are TCF7, BCL1 IB, and MYB.

191. The combination of gRNAs of claim 182, wherein the LCD genes are TCF7, MYB, and RUNX3.

192. A Cas-guide RNA (gRNA) combination comprising:(a) a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof fused to a transcriptional activation domain; and(b) a combination of gRNAs of any of claims 10 l-142e.

193. A Cas-guide RNA (gRNA) combination comprising:(a) a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof fused to a transcriptional activation domain; and(b) one or more gRNAs of any of claims 89-100.

194. The Cas-gRNA combination of claim 192 or claim 193, wherein the Cas protein or variant thereof is a deactivated (dCas) protein.

195. The Cas-gRNA combination of claim 194, wherein the dCas protein lacks nuclease activity.

196. The Cas-gRNA combination of claim 194 or claim 195, wherein the dCas protein is a dCas9 protein.

197. The Cas-gRNA combination of claim 196, wherein the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein.

198. The Cas-gRNA combination of claim 197, wherein the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 88.

199. The Cas-gRNA combination of claim 197 or claim 198, wherein the dSaCas9 protein comprises the sequence set forth in SEQ ID NO: 89, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

200. The Cas-gRNA combination of any of claims 197-199, wherein the dSaCas9 protein is set forth in SEQ ID NO: 89.

201. The Cas-gRNA combination of any of claims 194-196, wherein the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein.

202. The Cas-gRNA combination of claim 201, wherein the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO: 31.

203. The Cas-gRNA combination of claim 201 or claim 202, wherein the dSpCas9 protein comprises the sequence set forth in SEQ ID NO: 32, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

204. The Cas-gRNA combination of any of claims 201-203, wherein the dSpCas9 protein is set forth in SEQ ID NO: 32.

205. A polynucleotide encoding the DNA-targeting system of any of claims 1-117.

206. A polynucleotide encoding at least one DNA-targeting module of the DNA- targeting system of any of claims 1-117.

207. A polynucleotide encoding the gRNA of any of claims 118-135.

208. A polynucleotide encoding the combination of gRNAs of any of claims 136-186.

209. A polynucleotide encoding the Cas-gRNA combination of any of claims 187-204.

210. A plurality of polynucleotides encoding the DNA-targeting system of any of claims 1-117, the combination of gRNAs of any of claims 136-186, the Cas-gRNA combination of any of claims 187-204, or a portion or a component of any of the foregoing.

211. A plurality of polynucleotides comprising: one or more gRNAs of any of claims 118-135 or the combination of gRNAs of any of claims 136-186; and a polynucleotide encoding a fusion protein comprising a DNA-binding domain comprising a deactivated Cas (dCas) protein and at least one transcriptional activator effector domain.

212. The plurality of polynucleotides of claim 211, wherein the dCas protein is a dCas9 protein.

213. The plurality of polynucleotides of claim 211 or 212, wherein the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein.

214. The plurality of polynucleotides of claim 213, wherein the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO: 31.

215. The plurality of polynucleotides of claim 213 or claim 214, wherein the dSpCas9 protein comprises the sequence set forth in SEQ ID NO: 32, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

216. The plurality of polynucleotides of any of claims 213-215, wherein the dSpCas9 protein is set forth in SEQ ID NO: 32.

217. The plurality of polynucleotides of any of claims 211-216, wherein the at least one transcriptional activator effector domain comprises at least one VP 16 domain, and / or a VP 16 tetramer (“VP64”) or a variant thereof.

218. The plurality of polynucleotides of any of claims 211-217, wherein the at least one transcriptional activator effector domain comprises a VP64 domain or a variant or portion thereof that exhibits transcriptional activation activity.

219. The plurality of polynucleotides of any of claims 211-218, wherein the at least one transcriptional activator effector domain is VP64.

220. The plurality of polynucleotides of claim 219, wherein the VP64 is positioned N- terminal and / or C-terminal to the DNA-binding domain.

221. The plurality of polynucleotides of any of claims 211-220, wherein the at least one transcriptional activator effector domain comprises two copies of VP64.

222. The plurality of polynucleotides of any of claims 211-221, wherein the at least one transcriptional activator effector domain comprises the amino acid sequence set forth in SEQ ID NO: 28, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 28.

223. The plurality of polynucleotides of any of claims 211-222, wherein the at least one transcriptional activator effector domain comprises the amino acid sequence set forth inSEQ ID NO: 30, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 30.

224. The plurality of polynucleotides of any of claims 211-223, wherein the fusion protein comprises the sequence set forth in SEQ ID NO: 26, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

225. The plurality of polynucleotides of any of claims 211-224, wherein the polynucleotide encoding the fusion protein is an mRNA.

226. A plurality of polynucleotides encoding the epigenetic-modifying DNA-targeting system of any of claims 1-117.

227. A plurality of polynucleotides encoding at least one DNA-targeting module of the epigenetic-modifying DNA-targeting system of any of claims 1-117.

228. A plurality of polynucleotides encoding the fusion protein and the at least first gRNA and second gRNA of the epigenetic -modifying DNA-targeting system of any of claims 11-117.

229. A vector comprising the polynucleotide of any of claims 205-209 or the plurality of polynucleotides of any of claims 210-228.

230. The vector of claim 229 that is a viral vector.

231. The vector of claim 229 that is a lipid nanoparticle.

232. A pharmaceutical composition comprising the DNA-targeting system of any of claims 1-117, the Cas-gRNA combination of any of claims 192-204, the polynucleotide of any of claims 205-209, the plurality of polynucleotides of any of claims 210-228, or the vector of any of claims 229-231.

233. The pharmaceutical composition of claim 232 comprising a pharmaceutically acceptable excipient.

234. A method of differentiating a population of hematopoietic progenitor cells (HPCs) to a differentiated population of cells, the method comprising introducing the DNA- targeting system of any one of claims 1-117, the Cas-gRNA combination of any of claims 192- 204, the polynucleotide of any of claims 205-209, the plurality of polynucleotides of any of claims 210-228, the vector of any of claims 229-231, or a combination thereof, into a population of HPCs, and culturing the HPCs under conditions for their differentiation.

235. A method of differentiating a population of hematopoietic progenitor cells (HPCs) to a differentiated population of cells, the method comprising introducing the pharmaceutical composition of claim 232 or claim 233 into a population of hematopoietic progenitor cells (HPCs) and culturing the HPCs under conditions for their differentiation.

236. The method of claim 234 or claim 235, wherein the population of HPCs are induced hematopoietic progenitor cells (iHPCs).

237. The method of claim 234 or claim 235, wherein the population of HPCs are primary hematopoietic progenitor cells.

238. The method of any of claims 234-237, wherein the HPCs comprise cells engineered with a recombinant receptor, optionally a chimeric antigen receptor.

239. The method of any of claims 234-238, wherein the differentiated cells comprise cells that express a recombinant receptor, optionally a chimeric antigen receptor.

240. A population of differentiated cells produced by the method of any of claims 234- 239.

241. The method of any of claims 234-239, wherein cells of the differentiated population of cells are lymphoid progenitor cells.

242. The method of claim 241, wherein the lymphoid progenitor cells are induced common lymphoid progenitor cells (iCLPs).

243. The method of claim 241 or claim 242, wherein the lymphoid progenitor cells are CD5+ cells.

244. The method of any of claims 241-243, wherein the lymphoid progenitor cells have increased CD5 expression relative to the population of HPCs.

245. The method of any of claims 241-244, wherein the lymphoid progenitor cells are CD7+ cells.

246. The method of any of claims 241-245, wherein the lymphoid progenitor cells have increased CD7 expression relative to the population of HPCs.

247. The method of any of claims 241-246, wherein the lymphoid progenitor cells are CD5+CD7+ cells.

248. The method of any of claims 241-247, wherein the lymphoid progenitor cells have increased CD5 and CD7 expression relative to the population of HPCs.

249. The method of any of claims 241-248, wherein the lymphoid progenitor cells are CD45RA+ cells.

250. The method of any of claims 241-249, wherein the lymphoid progenitor cells have increased CD45RA+ expression relative to the population of HPCs.

251. The method of any of claims 241-250, wherein the lymphoid progenitor cells areCD117-mid cells.

252. The method of any of claims 241-251, wherein the lymphoid progenitor cells have increased CD117 expression relative to the population of HPCs.

253. The method of any of claims 241-252, wherein the lymphoid progenitor cells are CD34- cells.

254. The method of any of claims 241-253, wherein the lymphoid progenitor cells have decreased CD34 expression relative to the population of HPCs.

255. The method of any of claims 241-254, wherein the lymphoid progenitor cells are CD123- cells.

256. The method of any of claims 241-255, wherein the lymphoid progenitor cells have decreased CD123 expression relative to a population of monocytes.

257. The method of any of claims 241-256, wherein the lymphoid progenitor cells are CD14- cells.

258. The method of any of claims 241-257, wherein the lymphoid progenitor cells have decreased CD14 expression relative to a population of monocytes.

259. The method of any of claims 241-258, wherein the lymphoid progenitor cells are CD56- cells.

260. The method of any of claims 241-259, wherein the lymphoid progenitor cells have decreased CD56 expression relative to a population of lymphocytes.

261. The method of claims 241-260, wherein the lymphoid progenitor cells express a recombinant receptor, optionally a chimeric antigen receptor.

262. A population of lymphoid progenitor cells produced by the method of any of claims 241-261.

263. The method of any of claims 234-239, wherein cells of the differentiated population of cells are lymphoid cells (LCs)264. A method of generating lymphoid cells (LCs), the method comprising culturing the population of lymphoid progenitor cells produced by the method of any of claims 241-261 or the population of lymphoid progenitor cells of claim 262 under conditions to differentiate cells of the population to lymphoid cells (LCs) to produce a population comprising LCs.

265. The method of claim 263 or claim 264, wherein the differentiated population of cells are lymphoid cells (LCs).

266. The method of any of claims 263-265, wherein the LCs are induced Natural Killer (iNK) cells.

267. The method of claim 266, wherein the iNK cells are CD56+CD3- cells.

268. The method of claim 266 or claim 267, wherein the iNK cells are further characterized by one or more of the following: DNAM1+, NKG2D+, NKP30+ and / or CD16+.

269. A method of generating induced T (iT) cell progenitors, the method comprising contacting the population of lymphoid progenitor cells produced by the method of any of claims 241-261 or the population of lymphoid progenitor cells of claim 262 with at least one exogenous ligand comprising exogenous DLL4 ligand, optionally wherein the exogenous DLL4 ligand is coated on a substrate (e.g. well of a plate).

270. The method of 269, wherein the at least one exogenous ligand further comprises exogenous retronectin, optionally wherein the exogenous retronectin is coated on the substrate (e.g., well of the plate).

271. The method of claim 269 or claim 270, wherein the iT cell progenitors are double positive CD4+ / CD8+ cells.

272. A population of iT cell progenitors produced by the method of any of claims 269-271.

273. A method of generating iT cells, the method comprising culturing the population of iT cell progenitors produced by the method of any of claims 269-271 or the population of lymphoid progenitor cells of claim 272 under conditions to differentiate cells of the population to iT cell progenitors to produce a population comprising iT cells.

274. The method of 273, wherein the iT cells are characterized by one or more of the following: CD2+, CD3+, CD4+, and / or CD8b+.

275. The method of claim 273 or claim 274, wherein the iT cells are CD3+.

276. The method of claim 273-275, wherein the iT cells comprise T cells that are CD4+ or CD8+.

277. The method of claim 273-276, wherein the iT cells are CD8+ single positive cells.

278. The method of claims 273-277, wherein the iT cells comprise T cells that are CD8b+ cells.

279. The method of claims 273-278, wherein the iT cells comprise T cells that are CD8b+ cells.

280. The method of claim 273-279, wherein the iT cells are CD8b+ / CD8a+.

281. The method of any of claims 263-280, wherein the LCs, iT cell progenitors or iT cells express a recombinant receptor, optionally a chimeric antigen receptor.

282. A population of lymphoid cells, iT cell progenitors or iTcells produced by the method of any of claims 263-281.

283. The method of any of claims 234-239, 241-261, 263-271 and 273-281, that is carried out in vitro or ex vivo.

284. The method of any of claims 234-239, 241-261, 263-271, 273-281 and 283, wherein the HPCs are human HPCs.

285. The method of any of claims 234-239, 241-261, 263-271, 273-281, 283 and 284, wherein the introducing is by transient delivery into the population of HPCs.

286. The method of claim 285, wherein the transient delivery comprises electroporation, transfection, or transduction.

287. The method of any of claims 234-239, 241-261, 263-271, 273-281, and 283-286, wherein the introducing increases transcription of DLL4 and VCAM1.

288. The method of claim 287, wherein the introducing also increases transcription of the one or more lymphoid cell differentiation (LCD) genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL11B.

289. A composition comprising the the population of the population of differentiated cells of claim 240, the population of lymphoid progenitors of claim 262, the population of iT cell progenitors of claim 272, or the population of lymphoid cells of claim 282, optionally wherein the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient.

290. A method of treating a disease or condition in a subject, the method comprising administering to the subject the population of the population of differentiated cells of claim 240,the population of lymphoid progenitors of claim 262, the population of iT cell progenitors of claim 272, or the population of lymphoid cells of claim 282.

291. A method of treating a disease or condition in a subject, the method comprising administering to the subject the composition or pharmaceutical composition of claim 289.

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